Journal: Cell reports

Article Title: Dendritic distribution of autophagosomes underlies pathway-selective induction of LTD

doi: 10.1016/j.celrep.2023.112898

Figure Lengend Snippet:

Article Snippet: Horse polyclonal anti-mouse-HRP linked , Cell signaling Technology , Cat#7076; RRID: AB_330924.

Techniques: Virus, Recombinant, Sequencing, Plasmid Preparation, Software

Journal: bioRxiv

Article Title: HIF-1α-mediated mitochondrial-glycolytic reprogramming controls the transition of precursor to terminally exhausted T cells

doi: 10.1101/2023.08.31.555662

Figure Lengend Snippet: (a) Volcano plot of differential metabolite concentrations of in vitro activated WT and mPiC-deficient ( Slc25a3 fl/fl Cd4 Cre ) T cells analyzed by untargeted liquid chromatography and mass spectrometry (LC/MS); 4 biological replicates per group. (b) Analysis of NADPH/NADP + ratios in WT and mPiC-deficient T cells using LC/MS; 4 biological replicates per group. (c) Gene set enrichment analysis (GSEA) of the reactive oxygen species (ROS) pathway gene signature in WT and mPiC-deficient T cells. (d) Heatmap analysis of selected genes involved in ROS detoxification. (e) Flow cytometric analysis of cellular (CellROX) and mitochondrial ROS (MitoSox) in WT and mPiC-deficient T cells; means ± SEM of 5 mice. (f) Scavenging of ROS by N-acetylcysteine (NAC) prevents exhaustion of mPiC-deficient T cells. Flow cytometric analyses of CellRox, PD-1, Tim-3 and granzyme A expression in WT and mPiC-deficient T cells treated with NAC; means ± SEM of 4-8 mice. (g) Analysis of polyfunctional TNFα, IFNψ and IL-2 expression after PMA/iono restimulation of mPiC-deficient T cells treated with or without NAC; means ± SEM of 5 mice. (h) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis using bulk RNA sequencing data of WT and mPiC-deficient T cells. (i) Upstream transcription factor prediction analysis using differentially expressed genes (DEGs) between WT and mPiC-deficient T cells. (j) Analysis of Hif1a gene expression in WT and mPiC-deficient T cells by RNA sequencing; 3 biological replicates per group. (k) Flow cytometric analysis of HIF-1α protein expression in WT, mPiC- and HIF-1α-deficient T cells; means ± SEM of 3 mice. (l, m) Differentiation of WT, mPiC-deficient ( Slc25a3 fl/fl Cd4 Cre ) and mPiC/HIF-1α double-deficient ( Slc25a3 fl/fl Hif1a fl/fl Cd4 Cre ) T cells in vitro . Representative flow cytometric analysis (l) and quantification of PD-1, Tim-3, granzyme A, Lag3 and Ly108 expression on WT, mPiC-deficient and mPiC/HIF-1α double-deficient T cells (m); means ± SEM of 2-7 mice. *, p<0.05; **, p<0.01; ***, p<0.001 by unpaired Student’s t-test in (b), (e), (f), (g), (k) and (m).

Article Snippet: To measure HIF-1α protein expression, stimulated T cells were fixed with the Foxp3/TF staining buffer set (eBioscience) for 30 min at 4°C and stained intranuclearly with rabbit polyclonal anti-mouse HIF-1α (Cell Signaling) o/n in permeabilization buffer (eBioscience).

Techniques: In Vitro, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Expressing, RNA Sequencing Assay

Journal: bioRxiv

Article Title: HIF-1α-mediated mitochondrial-glycolytic reprogramming controls the transition of precursor to terminally exhausted T cells

doi: 10.1101/2023.08.31.555662

Figure Lengend Snippet: (a) Adoptive co-transfer of naïve tdTomato + GFP + WT and tdTomato + HIF-1α-deficient ( Hif1a fl/fl Cd4 Cre ) P14 T cells into C57BL/6 mice before chronic infection with LCMV clone 13 (LCMV CL ). 21 days post infection (d.p.i.), donor P14 WT and HIF-1α-deficient T cells were FACS sorted, barcoded and multiplexed in a 1:1 ratio and and subjected to single-cell (sc) RNA sequencing. Uniform manifold approximation and projection (UMAP) visualization of ∼ 8.400 non-proliferating ( Mki67 low/negative) T cells identified four clusters. Prediction of developmental trajectories using slingshot analysis; colour-coding according to pseudotime. (b) Normalised gene expression of Tcf7 , Slamf6 (Ly108), Sell (CD62L), Havcr2 (Tim-3), Cxcr6 and Gzma projected onto UMAP clusters. (c) Dot plot analysis of selected cluster markers representing Tpex and Tex cell subsets; color intensity and dot size represent z-score mean expression and percentage of cells expressing the gene, respectively. (d) Relative contribution of individual UMAP clusters in WT and Hif-1α-deficient P14 T cells. (e) Volcano plot of differentially expressed genes (DEGs) between WT and HIF-1α-deficient P14 T cells using scRNA sequencing. (f) Adoptive co-transfer of naïve GFP + WT and tdTomato + HIF-1α-deficient P14 T cells into C57BL/6 mice before chronic infection with LCMV CL . Flow cytometric analysis of Tpex and Tex cells in spleen and LNs of the host mice was performed 14 d.p.i.; n = 5-12 mice. *, p<0.05; ***, p<0.001 by paired Student’s t-test in (f).

Article Snippet: To measure HIF-1α protein expression, stimulated T cells were fixed with the Foxp3/TF staining buffer set (eBioscience) for 30 min at 4°C and stained intranuclearly with rabbit polyclonal anti-mouse HIF-1α (Cell Signaling) o/n in permeabilization buffer (eBioscience).

Techniques: Infection, RNA Sequencing Assay, Expressing, Sequencing

Journal: bioRxiv

Article Title: HIF-1α-mediated mitochondrial-glycolytic reprogramming controls the transition of precursor to terminally exhausted T cells

doi: 10.1101/2023.08.31.555662

Figure Lengend Snippet: (a) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis using single cell (sc) RNA sequencing of WT and HIF-1α-deficient T cells. (b, c) Violin plots displaying Pfkl, Aldoa, Tpi1, Gapdh , Eno1 and Pkm gene expression in Tpex (b) and Tex cells (c) of WT and HIF-1α-deficient ( Hif1a fl/fl Cd4 Cre ) P14 T cells analyzed by scRNA sequencing. (d) Differentiation of WT and HIF-1α-deficient T cells under hypoxia in vitro , means ± SEM of 6 mice. (e, f) Analyses of glycolytic proton efflux rate (glycoPER) (e) and oxygen consumption rate (OCR) (f) in WT and HIF-1α-deficient T cells using a Seahorse extracellular flux analyzer; means ± SEM of 3 mice. (g) Relative contribution of glycolysis and mitochondrial respiration to cellular ATP production in WT and HIF-1α-deficient T cells; means ± SEM of 3 mice. (h, i) Inhibition of glycolysis sustains the stemness of virus-specific T cells. (h) Adoptive co-transfer of 2-DG pre-treated (tdTomato + GFP + ) and control (GFP + ) P14 T cells into C57BL/6 mice after chronic infection with LCMV CL . Flow cytometric analysis of Tpex and Tex cells among donor P14 cells in spleen and LNs 10 d.p.i.; n = 6 mice. (i) Analysis of polyfunctional TNFα, IFNψ and IL-2 expression after PMA/iono restimulation of 2-DG-treated and control P14 T cells 10 days after co-transfer into chronically infected mice; means ± SEM of 6 mice. (j-l) 2-DG treatment during manufacturing augments the antitumor efficacy of CAR T cells. (j) 1×10 anti-hCD19 CAR T cells treated with or without 2-DG for 24 h were adoptively transferred into Rag1 −/– mice 7 days after MC38 hCD tumor inoculation. (k, l) Analysis of tumor growth (k) and cumulative survival (l) of MC38 hCD -bearing host mice after transfer of 2-DG or control-treated anti-hCD19 CAR T cells; means ± SEM of 9-11 mice. *, p<0.05; **, p<0.01; ***, p<0.001 by unpaired and paired Student’s t-test in (b-d) and (h). *, p<0.05 and **, p<0.05 in (k) and (l) by 2-way ANOVA and Mantel-Cox test, respectively.

Article Snippet: To measure HIF-1α protein expression, stimulated T cells were fixed with the Foxp3/TF staining buffer set (eBioscience) for 30 min at 4°C and stained intranuclearly with rabbit polyclonal anti-mouse HIF-1α (Cell Signaling) o/n in permeabilization buffer (eBioscience).

Techniques: RNA Sequencing Assay, Expressing, Sequencing, In Vitro, Inhibition, Virus, Infection