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. 2009 Jul;5(5):625-35.
doi: 10.4161/auto.5.5.8133. Epub 2009 Jul 10.

Identification of Atg5-dependent transcriptional changes and increases in mitochondrial mass in Atg5-deficient T lymphocytes

Linda M Stephenson  1 , Brian C Miller, Aylwin Ng, Jason Eisenberg, Zijiang Zhao, Ken Cadwell, Daniel B Graham, Noboru N Mizushima, Ramnik Xavier, Herbert W Virgin, Wojciech Swat
Affiliations

Identification of Atg5-dependent transcriptional changes and increases in mitochondrial mass in Atg5-deficient T lymphocytes

Linda M Stephenson et al. Autophagy. 2009 Jul.

Abstract

Autophagy is implicated in many functions of mammalian cells such as organelle recycling, survival and differentiation, and is essential for the maintenance of T and B lymphocytes. Here, we demonstrate that autophagy is a constitutive process during T cell development. Deletion of the essential autophagy genes Atg5 or Atg7 in T cells resulted in decreased thymocyte and peripheral T cell numbers, and Atg5-deficient T cells had a decrease in cell survival. We employed functional-genetic and integrative computational analyses to elucidate specific functions of the autophagic process in developing T-lineage lymphocytes. Our whole-genome transcriptional profiling identified a set of 699 genes differentially expressed in Atg5-deficient and Atg5-sufficient thymocytes (Atg5-dependent gene set). Strikingly, the Atg5-dependent gene set was dramatically enriched in genes encoding proteins associated with the mitochondrion. In support of a role for autophagy in mitochondrial maintenance in T lineage cells, the deletion of Atg5 led to increased mitochondrial mass in peripheral T cells. We also observed a correlation between mitochondrial mass and Annexin-V staining in peripheral T cells. We propose that autophagy is critical for mitochondrial maintenance and T cell survival. We speculate that, similar to its role in yeast or mammalian liver cells, autophagy is required in T cells for the removal of damaged or aging mitochondria and that this contributes to the cell death of autophagy-deficient T cells.

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Figures

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Figure 1. Constitutive autophagy in all subsets of wild-type T cells. (A) Lysates from FACS-sorted C57BL/6 thymus cells and MACS-bead sorted peripheral T cells were probed with antibodies against LC3 and β-actin. Representative blot from four independent experiments shown. (B) Lysates from C57BL/6 thymocytes cultured for 4 hours with the indicated concentrations of chloroquine were probed with antibodies against LC3 and β-actin. Representative blot from three independent experiments shown. (C) Lysates from Atg5+/+ or Atg5−/− chimeras or C57BL/6 control thymocytes were probed with antibodies against ATG5, LC3 or β-actin. Representative blot from two independent experiments shown. (D) Lysates from Atg5+/+ and Atg5−/− SV40-transformed murine embryonic fibroblasts and from Atg5F/F Cre+ and Atg5F/F Cre− thymocytes were probed with antibodies against ATG5 and β-actin. Representative blot from three independent experiments shown. (E) Lysates from Atg7F/F Cre+ and Atg7F/F Cre− thymocytes were probed with antibodies against ATG7 and β-actin. Representative blot from two independent experiments shown.
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Figure 2. Loss of T cells in Atg5−/− chimeric, Atg5F/F Cre+ and Atg7F/F Cre+ mice. Single-cell suspensions of thymocytes, splenocytes and lymph node cells were analyzed by flow cytometry as described in Materials and Methods. Cells were gated by forward and side scatter on lymphocyte populations. Shown is one representative experiment from n ≥ 3 experiments, with at least seven mice of each genotype analyzed.
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Figure 3.Atg5 is required for the proper regulation of mitochondrial-associated gene sets. (A) Microarray analysis showing genes differentially expressed (p < 0.05), of which 259 genes were induced and 440 downregulated in Atg5−/− thymocytes compared to wild-type thymocytes. Expression values for each probeset were z-score-transformed across all arrays and their intensities above and below the mean are represented on the heatmap by red and green colors respectively, as shown on the color bar. Genes were hierarchically clustered using Cluster 3.0 and visualized with Java TreeView. Tick markings highlight 64 differentially expressed mitochondrial genes, whose expression profiles are displayed by the heatmap on the right. (B) Differentially expressed genes were classified into gene ontology (GO) cellular component categories. Categories assigned with at least 3 genes are displayed as a pie chart, with enrichment p-values shown in brackets alongside each category. The bar chart displays enrichment p-values as negative log-transformed values, which reveals a dramatic enrichment of mitochondria- and nucleus-associated genes. (C) Construction of human protein-protein interaction (interolog) network. Differentially expressed genes were mapped onto human orthologs for which interaction data was available. Circles representing upregulated components are colored red and downregulated components green. Solid lines denote protein-protein interactions. Mitochondrion-anchored subnetworks (highlighted by the yellow region) emerged when extending connections by an additional degree of separation, capturing components of interest (blue circles) in the functional neighborhood of mitochondrial-related genes that were found to be differentially expressed. Network clusters containing connections between at least 3 components are displayed. (D) Diagrammatic mitochondrion representation showing key differentially expressed gene products participating in mitochondria-associated processes. Red and green ovals denote differentially up and downregulated components respectively. Functional associations with biological processes are represented by dashed lines.
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Figure 4. A higher percentage of T cells are CD44high, CD62Llow and Annexin-V+ in Atg5−/− chimeras and Atg5F/F Cre+ mice compared to controls. (A and B) Flow cytometric analysis of lymph node T lymphocytes from Atg5+/+ or Atg5−/− chimeric mice (A) or Atg5F/F Cre− and Atg5F/F Cre+ mice (B). Cells were gated by forward and side scatter for lymphocytes, then gated on CD4+ or CD8+ cells. One representative experiment shown of at least five independent experiments. (C and D) Lymph node T cells from Atg5+/+ or Atg5−/− chimeric mice (C) or Atg5F/F Cre− or Atg5F/F Cre+ mice (D) were stained with Annexin-V and antibodies against CD4, CD8 and CD62L. Cells were gated by forward and side scatter for lymphocytes, then gated on CD4+ or CD8+ cells. Data pooled from at least four independent experiments. (*p < 0.05, **p < 0.005, ***p < 0.0005, n.s. = not statistically significant).
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Figure 5. Proliferation defect in Atg5-deficient T lymphocytes. Purified CD62Lhigh T cells from Atg5+/+ or Atg5−/− chimeric mice were either untreated or stimulated with anti-CD3 (1 µg/mL) and anti-CD28 (1 µg/mL) and analyzed 40 hours later for T cell blasts (left panels) or loaded with CFSE, stimulated as above, and analyzed 72 hours later (right panel). Shown is one representative experiment of three. CD4+ cells shown.
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Figure 6.Atg5-deficient T lymphocytes have an increase in mitochondrial mass. (A) Splenocytes from Atg5F/F Cre+ and Atg5F/F Cre− mice were loaded with MitoTracker green, stained with antibodies against CD4 or CD8, and analyzed by flow cytometry. Analysis was performed on CD4+ or CD8+ splenocytes without gating by forward and side scatter. Shown are representative FACS plots of at least fifteen mice of each genotype and the quantation of the mean fluorescence intensity (MFI) of Mitotracker staining from one of three independent experiments. (B) Splenocytes from Atg5F/F Cre+ and Atg5F/F Cre− mice were stained with CD4, CD8, Mitotracker green, and Annexin-V. Representative FACS plots and quantitation of the percentage of Annexin-V+ cells from the Mitotrackerhigh and Mitotrackerlow gates are shown. Analysis was performed on CD4+ or CD8+ splenocytes without gating by forward and side scatter. Data pooled from three independent experiments. (*p < 0.05, **p < 0.005, ***p < 0.0005, n.s. = not statistically significant).
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