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Clearance in Mature T LymphocytesAutophagy Is Essential for Mitochondrial

HeHeather H. Pua, Jian Guo, Masaaki Komatsu and You-Wen

http://www.jimmunol.org/content/182/7/4046doi: 10.4049/jimmunol.0801143

2009; 182:4046-4055; ;J Immunol 

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Autophagy Is Essential for Mitochondrial Clearance in MatureT Lymphocytes1

Heather H. Pua,* Jian Guo,* Masaaki Komatsu,† and You-Wen He2*

Macroautophagy plays an important role in the regulation of cell survival, metabolism, and the lysosomal degradation ofcytoplasmic material. In the immune system, autophagy contributes to the clearance of intracellular pathogens, MHCIIcross-presentation of endogenous Ags, as well as cell survival. We and others have demonstrated that autophagy occurs inT lymphocytes and contributes to the regulation of their cellular function, including survival and proliferation. Here we showthat the essential autophagy gene Atg7 is required in a cell-intrinsic manner for the survival of mature primary T lympho-cytes. We also find that mitochondrial content is developmentally regulated in T but not in B cells, with exit from the thymusmarking a transition from high mitochondrial content in thymocytes to lower mitochondrial content in mature T cells.Macroautophagy has been proposed to play an important role in the clearance of intracellular organelles, and autophagy-deficient mature T cells fail to reduce their mitochondrial content in vivo. Consistent with alterations in mitochondrialcontent, autophagy-deficient T cells have increased reactive oxygen species production as well as an imbalance in pro- andantiapoptotic protein expression. With much recent interest in the possibility of autophagy-dependent developmentallyprogrammed clearance of organelles in lens epithelial cells and erythrocytes, our data demonstrate that autophagy may havea physiologically significant role in the clearance of superfluous mitochondria in T lymphocytes as part of normal T cellhomeostasis. The Journal of Immunology, 2009, 182: 4046 – 4055.

M acroautophagy (hereafter referred to as autophagy) isa well-conserved catabolic process in eukaryoticcells characterized by the formation of double-

membrane vesicles 0.5–1.5 �m in diameter in the cytoplasm ofcells termed autophagosomes (1, 2). Autophagosomes arisethrough the elongation of cup-shaped isolation membranes thatform spherical vesicles before fusing with lysosomes to becomedegradative compartments. Mature autophagosomes encase bothcytosol as well as organelles, consistent with the early character-ization of autophagy as a major pathway for protein degradationduring periods of starvation (3). Autophagy induction is regulatedby the activity of class III PI3K in complex with the essentialautophagy gene Beclin-1 (yeast Atg6). Two ubiquitin-like conju-gation pathways involving the autophagy genes Atg3, Atg5, Atg7,microtubule-associated protein light chain 3 (LC3, yeast Atg8),Atg10, and Atg12 are also required for the formation of autopha-gosomes (1, 4).

The induction of autophagy generally executes two complemen-tary functions in eukaryotes that include the recycling of usefulmetabolic substrates as well as the removal of cytoplasmic mate-rial (1, 5, 6). The digestion of intracellular proteins to generatemetabolic substrates is essential for cell survival during periods of

starvation or growth factor deprivation. This is best demonstratedin mice lacking Atg5 or Atg7, which both succumb to starvation asneonates before robust suckling (7, 8). Importantly, it has also beenshown that the survival of an IL-3-dependent hematopoietic cellline during cytokine deprivation depends on autophagosome for-mation to provide metabolic substrates (9). Additionally, autoph-agy-mediated clearance of toxic cytoplasmic materials is criticalfor neural cell survival, as conditional deletion of Atg5 or Atg7 inneurons leads to the accumulation of ubiquitin-positive inclusionsand widespread neural cell death (10, 11).

Mounting evidence implicates autophagy in mitochondrialremoval (often designated “mitophagy”) in both mammalianand yeast cells (12). Mitochondria have been found within ves-icles that possess double or multiple membranes in rat hepato-cytes (13), hamster erythroid cells (14), and yeast (15). Mod-ulating autophagy by rapamycin treatment or expression ofGAPDH affects mitochondrial mass within cells (16, 17). Mi-tochondrial permeability transition triggers autophagy-medi-ated mitochondrial degradation (18, 19). Mitophagy is also in-duced by cytotoxic agents in the presence of caspase inhibitors(20). In vivo, there has been much interest in the physiologicrole of autophagy in mitochondrial clearance during lens anderythroid differentiation (14, 21–24).

Within immunology, there is an expanding role for autophagy inboth the innate and adaptive immune system (25, 26). Autophagycontributes to the clearance of intracellular pathogens (27–31) aswell as the MHCII cross-presentation of endogenous Ags (32, 33).We and others have also demonstrated a role for autophagy inlymphocytes. In T lymphocytes, double-membrane autophago-somes form in both human and murine T cells and can be inducedin TCR-stimulated proliferating cells in vitro (34–37). Althoughthe functional consequences of autophagosome formation in lym-phocytes are not well understood, genetic and molecular studieshave demonstrated a complex role for autophagy in T cell survivaland function. Loss of the essential autophagy gene Atg5 impairsthe survival and proliferation of mature T cells in vivo (35), while

*Department of Immunology, Duke University Medical Center, Durham, NC 27710;and †PRESTO (Precursory Research for Embryonic Science and Technology), JapanScience and Technology Corporation, Kawaguchi and Laboratory of Frontier Science,Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo, Japan

Received for publication April 8, 2008. Accepted for publication January 26, 2009.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by National Institutes of Health Grant AI-073947.2 Address correspondence and reprint requests to Dr. You-Wen He, Department ofImmunology, Duke University Medical Center, Box 3010, Durham, NC 27710.E-mail address: he000004@mc.duke.edu

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

The Journal of Immunology

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induction of autophagy by the HIV envelope glycoprotein inducesdeath in target lymphocytes (36). In B lymphocytes, autophagy isrequired for survival of developing pre-B cells as well as matureB1 B cells (38).

In this paper, we demonstrate that the essential autophagy geneAtg7 is required for mature T cell survival. We show that mito-chondrial content is developmentally regulated in T lymphocytesand that autophagy is critical for the clearance of mitochondrialcontent in mature T cells.

Materials and MethodsAnimals

Atg7-floxed and Atg5-deficient mice were generated and characterized pre-viously (7, 8). To generate mice with Atg7-deficient T lymphocytes, Atg7-floxed mice were crossed to Lck-Cre transgenic mice (39) (The JacksonLaboratory). Atg7 genomic deletion was assessed by PCR detection of theAtg7-floxed allele (forward, TGA GAC ATG GCC TGA AGA AAC CCA;reverse, ATG CTG CAG GAC AGA GAC CAT CA) and the wild-typegenomic locus (forward, TTA CAG TCG GCC AGG CTG AC; reverse,CCT GGG CTG CCA GAA TTT CTC) in FACS-sorted thymocytes andmature T cells isolated with DNeasy spin kits (Qiagen). Atg5 chimeraswere generated by transferring fetal liver cells into lethally irradiated con-genic recipient mice as described (35). All animal usage has been approvedby the Duke University Institutional Animal Care and Use Committee.

Western blot

Total thymocytes, EasySep-enriched lymph node (LN)3 T cells (StemCellTechnologies), and sorted TCR��CD4� and TCR��CD8� T cells fromLNs were lysed in 50 mM Tris (pH 6.8), 10% glycerol, 2% SDS, and 100mM DTT. Membranes were blotted with Abs recognizing Atg7 (ProSci),Atg5 (Proteintech Group), cytochrome C (BD Pharmingen), Tom20 (SantaCruz Biotechnology), apoptosis-inducing factor (AIF; Cell Signaling Tech-nology), mtHsp70 (Affinity BioReagents), Bcl-2 (BD Pharmingen), Bcl-xL

(BD Pharmingen), Mcl-1 (Rockland), Bak (Upstate), Bax (eBioscience),and actin (Santa Cruz Biotechnology). All blots except Bcl-2 were visu-alized with anti-rabbit Alexa 680 (eBioscience), anti-mouse Alexa680(eBioscience), and anti-goat Alexa 800 (Rockland) secondary Abs and readusing an infrared imaging system (LI-COR Bioscience). Bcl-2 was visu-alized with HRP-conjugated anti-Armenian hamster (Jackson Immuno-Research Laboratories) and Pico chemiluminescent substrate (Pierce).

Flow cytometry

Single-cell suspensions of thymus, spleen, and LNs were lysed of RBCand incubated with FcR blocker (2.4G2; eBioscience). Cells were main-tained in PBS with 2% FBS on ice throughout staining before analysis.Cells were stained with FITC, PE, PE-Cy5, allophycocyanin, and/orallophycocyanin-Cy7 anti-CD4, -CD8, -TCR�, -CD44, -CD62L,-CD25, -B220, and -CD45.2 (eBioscience, BioLegend, and BD Pharm-ingen). Cell events were collected on a FACScan or FACStar, and datawere analyzed using CellQuest (Becton Dickson) and FlowJo (TreeStar) software. Cell death was assayed using annexin V, 7-aminoacti-nomycin D, and propidium iodide staining (BD Pharmingen) or an ac-tivated caspase 9 detection kit (Immunochemistry Technologies). Tostain mitochondria, lymphocytes were incubated for 30 min at 37°Cwith 100 nM MitoTracker Green (Molecular Probes) in RPMI 1640complete medium before surface Ab staining. For intracellular stains,cells were fixed in 2% PFA and permeabilized with 0.2% saponin(Sigma-Aldrich) before staining with anti-Bcl-2 PE (BD Pharmingen)or anti-Mcl-1 (Rockland) followed by FITC anti-rabbit Abs (JacksonImmunoResearch Laboratories).

Transmission electron microscopy

Mature LN T cells after EasySep negative bead selection (StemCell Tech-nologies) were Ab stained and lightly fixed in 1% PFA. One to two millionTCR��CD8� T cells were sorted and fixed in a 4% glutaraldehyde 0.1 Msodium cacodylate buffer overnight. The samples were rinsed in 0.1 Mcacodylate buffer containing 7.5% sucrose three times for 15 min each andfixed in 1% osmium in cacodylate buffer for 1 h. After being washed threetimes in 0.11 M veronal acetate buffer for 15 min each, the samples were

incubated with 0.5% uranyl acetate in veronal acetate buffer for 1 h at roomtemperature. Specimens were then dehydrated in an ascending series ofethanol (35%, 70%, 95%, and two changes of 100%) for 10 min each,followed by two changes of propylene oxide for 5 min each. The sampleswere incubated with a 1:1 mixture of 100% resin and propylene oxide for1 h, followed by two changes of 100% resin, each for 30 min. Finally, thesamples were embedded in resin and polymerized at 60°C overnight. Thicksections (0.5 �m) were cut and stained with toluidine blue for light mi-croscopy selection of the appropriate area for ultrathin sections. Thin sec-tions (60–90 nm) were cut, mounted on copper grids, and poststained withuranyl acetate and lead citrate. Micrographs were taken with a Philips LS410 electron microscope. Images were analyzed using AxioVision software(Zeiss).

Reactive oxygen species (ROS) assay

To assay for ROS production, lymphocytes were incubated in 5 �M di-hydroethidium (Sigma-Aldrich) or CM-H2DCFDA (Molecular Probes) for1 h in RPMI 1640 complete medium at 37°C and subsequently analyzed byflow cytometry. ROS production was inhibited in vitro by the addition of150 �M manganese (III) tetrakis 4-benzoic acid (MnTBAP; Calbiochem),1 mM N-acetylcysteine (Sigma-Aldrich), 50 U/ml superoxide dismutase-polyethylene glycol (Sigma-Aldrich), or 50 U/ml catalase-polyethyleneglycol (Sigma-Aldrich).

Quantitative PCR

To determine mitochondrial DNA content in primary T cells, EasySep-enriched (StemCell Technologies) CD8� cells were sorted and DNA wasextracted using a DNeasy kit (Qiagen). Quantitative PCR was performedusing a LightCycler FastStart DNA Master SYBR Green I kit on an iCycleriQ real-time PCR detection system (Roche). Products from two sets ofmitochondrial DNA-specific primers (mtDNA F1, ACC ATT TGC AGACGC CAT AA; mtDNA R1, TGA AAT TGT TTG GGC TAC GG (40);mtDNA F2, GCC CCA GAT ATA GCA TTC CC; mtDNA R2, GTT CATCCT GTT CCT GCT CC (41); actin DNA F, TGT TCC CTT CCA CAGGGT GT; actin DNA R, TCC CAG TTG GTA ACA ATG CCA (41)) werenormalized to a genomic actin cell loading control and analyzed usingrelative expression software tool (REST) v2 software (42, 43).

ResultsSurvival defect in Atg7-deficient mature T cells

Previous work by our group (35) and others (34, 36, 37) has dem-onstrated that autophagosomes form in primary T lymphocytes.Additionally, we have also demonstrated that the autophagy geneAtg5 contributes critically to the homeostatic survival of mouse Tlymphocytes in vivo (35). As this autophagy protein also interactswith FADD (Fas-associated death domain protein) and Bcl-xL (44,45), Atg5 may regulate T cell survival through its role in autopha-gosome formation or through interactions with extrinsic or intrin-sic death pathways (46, 47). To determine whether the process ofautophagy contributes to mature T cell survival, we examined theT cell compartment in a second autophagy-deficient genetic modelsystem. We crossed Atg7f/f mice (8) with mice expressing the Tcell-specific Lck-Cre transgene (39). Consistent with the inductionof Lck-Cre expression at the CD4�CD8� double-negative (DN)stage of thymocyte development, we observed efficient genomicand protein deletion of Atg7 in double-positive (DP) and single-positive (SP) thymocytes as well as in mature CD4� and CD8� Tlymphocytes in Atg7f/fLck-Cre mice (Fig. 1A). Additionally, wefound impaired conjugation of Atg5 to Atg12 in both thymocytesand peripheral T cells lacking Atg7, an essential upstream enzymefor this autophagic pathway conjugation process (Fig. 1B). Thesedata demonstrate that Atg7 is efficiently deleted, and they are con-sistent with a functional deficiency in autophagy in our modelsystem.

To determine the role of autophagy in T cell development andfunction, we examined thymocytes and mature T cells in Atg7f/f-Lck-Cre and Atg7f/f littermate controls. Although thymocyte de-velopment appeared grossly normal in Atg7f/fLck-Cre mice (Fig.1C), pooled data from large numbers of mice showed a modest but

3 Abbreviations used in this paper: LN, lymph node; AIF, apoptosis-inducing factor;DN, double negative; DP, double positive; MFI, mean fluorescence intensity; ROS,reactive oxygen species; SP, single positive.

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statistically significant decrease in CD4� and CD8� SP thymo-cytes in these animals (Fig. 1E). In the spleen and lymph nodes,there was a dramatic reduction in the percentage of T cells in

Atg7f/fLck-Cre mice (Fig. 1C). Atg7f/fLck-Cre mice also demon-strated an increase in the relative percentage of CD44high

CD62Llow peripheral T cells (Fig. 1D). This phenotype is most

FIGURE 1. Impaired number of peripheral T cells in Atg7f/fLck-Cre mice. Detection of Atg7 genomic deletion by PCR (A) and Atg7 protein (B) byWestern blot in thymocytes and T cells from Atg7f/f and Atg7f/fLck-Cre mice. Atg7-dependent conjugation of Atg5 to Atg12 was also detected by anti-Atg5Western blot in Atg7f/f and Atg7f/fLck-Cre thymocytes and LN T cells. In both Atg7 and Atg5 Western blots, specific bands are marked with an arrow,and protein size is indicated. C, Flow cytometry analysis of Atg7f/f and Atg7f/fLck-Cre thymocytes, splenocytes, and LN T cells stained with CD4 and CD8Abs. Numbers indicate percentage of total live cells. D, Flow cytometry analysis of CD44, CD62L, CD25, and CD69 on mature T cells from LN of Atg7f/f

and Atg7f/fLck-Cre mice. Numbers indicate percentage of total CD4� or CD8� T cells. E, Total cell numbers of thymocyte subsets, CD4� T cells, CD8�

T cells, and B cells in Atg7f/f and Atg7f/fLck-Cre mice. Data pooled from 3 to 10 mice in three to five independent experiments (means � SD). �, p �

0.05; ��, p � 0.005; ���, p � 0.0005.

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consistent with lymphopenia, as markers of cell activation includ-ing CD69 and CD25 were not increased in these mice (Fig. 1D).Ultimately, this resulted in a 75% decrease in total naive T cellnumbers in peripheral Atg7-deficient T cells in secondary lym-phoid tissues (Fig. 1E). Finally, unlike Atg5�/� fetal liver chime-ras where total B lymphocyte numbers are decreased (35, 38),we observed no reduction in B cell numbers in our model sys-

tem consistent with the selective elimination of Atg7 in T lin-eage cells (Fig. 1E).

Although numerous factors including modestly reduced thymo-cyte numbers (Fig. 1E) and alterations in the proliferative capacityof autophagy-deficient T cells (38) may contribute to the defect inAtg7-deficient peripheral T cell homeostasis, mature T cells lack-ing this essential autophagy gene undergo enhanced apoptotic

FIGURE 2. Enhanced apoptotic death in matureAtg7f/fLck-Cre T cells. Flow cytometry analysis offreshly isolated Atg7f/f and Atg7f/fLck-Cre T cells forcell death. A, Percentage of dying (annexin V�7-AAD�) or dead (annexin V�7-AAD�) cells amonggated CD4� or CD8� T cells. B, Percentage of CD4� orCD8� T cells with active intracellular caspase 9 by flowcytometry.

FIGURE 3. Increased mitochondrial content in autophagy-deficient T cells. A, Flow cytometry analysis of MitoTracker Green mitochondrial staining innaive CD44lowAtg7f/f and Atg7f/fLck-Cre splenic T cells with MFI indicated. B, Mitochondrial content in Atg7-deficient peripheral lymphocytes asexpressed by percentage of MitoTracker MFIs relative to control cells. Data were derived from four to six mice in three independent experiments (errorbars, SD). C, MitoTracker and Tom20 colocalization in control and autophagy-deficient T cells. LN T cells were stained with MitoTracker Red andCD4-FITC, fixed, and permeabilized for intracellular staining with Tom20 and Alexa 350 anti-rabbit Ab. Confocal images were taken with the ZeissApoTome system using AxioVision software and inspect for colocalization of mitochondrial markers. Data are representative of two experiments. D,Coupled mitochondrial content in Atg7-deficient peripheral lymphocytes as expressed by percentage of potential-sensitive tetramethylrhodamine ethyl esterperchlorate (TMRE) MFIs relative to control cells. Data were derived from three mice from two independent experiments (error bars, SE). E, Mitochondrialcontent in donor and host lymphocytes in Atg5-deficient and control fetal liver chimeras expressed as percentage of MitoTracker MFIs relative to controlcells. Data were derived in four to six mice from three independent experiments (error bars, SD). �, p � 0.05 and ��, p � 0.005.

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death. Freshly isolated mature Atg7f/fLck-Cre T cells displayed 3-to 4-fold enhanced rates of apoptosis ex vivo as measured by an-nexin V surface staining (Fig. 2A). Consistent with enhancedapoptotic death, freshly isolated Atg7-deficient T cells alsodemonstrated enhanced caspase 9 activity by flow cytometry (Fig.2B). Taken together, these data demonstrate an important role forthe essential autophagy gene Atg7 in maintaining peripheral Tlymphocyte homeostasis. With previously published data fromAtg5-deficient chimeric mice, our results support a role for auto-phagy in promoting mature T cell survival.

Enhanced mitochondrial content in Atg7-deficient mature T cells

The above results suggest that autophagy may have cytoprotectivefunctions in T lymphocytes. Given the central role of mitochondriain lymphocyte apoptotic cell death (48) and the emerging role forautophagy in maintaining proper homeostasis of this organelle (6),we investigated the mitochondrial compartment in autophagy-de-ficient T cells. We first examined the mitochondria in Atg7f/f andAtg7f/fLck-Cre mature T cells using the relatively potential-inde-pendent mitochondria-specific vital dye MitoTracker Green (49).MitoTracker Green is a lipophilic thiol-reactive dye that selec-tively labels mitochondrial inner membrane and matrix (50) andhas been used for measuring mitochondrial content in hematopoi-etic cells (51–53). Analysis by flow cytometry revealed a50–150% increase in the MitoTracker Green staining of Atg7-

deficient CD4� and CD8� T lymphocytes in the spleen and lymphnodes (Fig. 3, A and B). As expected from this T cell-specificdeletion model, we observed no difference in MitoTracker Greenstaining of B220� B lymphocytes (Fig. 3B).

Additionally, we confirmed this difference in the CD44low naiveT cell subset since Atg7f/fLck-Cre mice have a relative increase inCD44highCD62Llow T cells consistent with a homeostatic responseto lymphopenia (Fig. 3, A and B). We also examined whetherMitoTracker Green staining measured by flow cytometry was spe-cific for mitochondria by immunofluorescent microscopy. In bothAtg7f/f and Atg7f/fLck-Cre T cells, MitoTracker stained a net-worked cytoplasmic structure that also stained positive for theouter mitochondrial membrane marker Tom20 (Fig. 3C). Finally,we observed an increase in staining by the mitochondrial po-tential- and volume-dependent dye TMRE in Atg7f/fLck-Cre Tcells when compared with Atg7f/f controls, consistent with anexpansion of the functional mitochondrial content in these cells(Fig. 3D).

We next examined mitochondrial content in mature T cells lack-ing Atg5. In Atg5�/� fetal liver chimeric mice, CD45.2� donor-derived CD4� and CD8� T cells from the spleen and lymph nodesdemonstrated dramatically enhanced MitoTracker Green staining(Fig. 3E). No difference in MitoTracker Green staining in residualhost T cells was observed in Atg5�/� and Atg5�/� chimeras (Fig.3E). Interestingly, Atg5�/� and control peripheral B lymphocytes

FIGURE 4. Developmentally regulated mitochon-drial content changes in T lymphocytes. A, Flow cytom-etry analysis of MitoTracker Green mitochondrial stain-ing in Atg7f/f and Atg7f/fLck-Cre thymocytes with MFIindicated. B and C, Mitochondrial content in Atg7 (B)or Atg5-deficient (C) thymocytes as expressed by per-centages of their MitoTracker MFIs relative to controlcells. Data were derived from four to eight mice in threeindependent experiments (error bars, SD). ���, p �

0.0005. D, Comparison of MitoTracker Green stainingbetween thymocytes and peripheral T cells by normal-izing Atg7f/f (filled symbols) and Atg7f/fLck-Cre (opensymbols) MFI to Atg7f/f DP fluorescence. Data fromthree to four mice from three independent experiments(error bars, SD). E, Mitochondrial content is unchangedduring B lymphocyte development. Comparison ofMitoTracker Green staining in developing bone marrowand peripheral wild-type B cells by normalizing MFI topro-B cell fluorescence. Mitochondrial content wasmeasured by flow cytometry in CD43�B220� pro-Bcells, CD43�B220low pre-B cells, CD43�B220high im-mature B cells, and B220� peripheral B cells in thespleen (Spl) and LN. Data are from three mice (errorbars, SD).

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had comparable MitoTracker Green staining, suggesting a T cell-specific role for autophagy in mitochondrial homeostasis withinthe lymphocyte compartment (Fig. 3E).

Regulated mitochondrial content changes in developing T cells

To determine whether developing autophagy-deficient T cellsalso contain enhanced mitochondrial content, we analyzed Mito-Tracker Green fluorescence in Atg7f/f and Atg7f/fLck-Cre thy-

mocytes. Although there was a significant increase in Mito-Tracker Green mean fluorescent intensity (MFI) in Atg7-deficient CD4� and CD8� SP thymocytes, the magnitude ofthis change (10 – 40%) was low when compared with matureperipheral Atg7-deficient CD4� and CD8� T cells (Fig. 4, Aand B). Additionally, we did not observe an increase in Mito-Tracker Green staining in Atg5�/� thymocytes when comparedwith Atg5�/� controls (Fig. 4C). The reason for this

FIGURE 5. Mitochondrial morphology and DNA content in Atg7-deficient T cells. A, Representative transmission electron micrograph of Atg7f/f andAtg7f/fLck-Cre CD8� T cell, with enlarged region showing mitochondrial morphology. B, Surface area of mitochondria in CD8� T cell cross-sections.Values were calculated after manually outlining mitochondria and using the measure tool in AxioVision software. �, p � 0.05. C, Mitochondrial DNAcontent in Atg7-deficient CD8� T cells. Quantitative PCR using two sets of primers specific for mitochondrial DNA was performed and normalized forinput using a control genomic locus in sorted peripheral CD8�CD44low T cells from Atg7�/� and Atg7-deficient mice. Mitochondrial DNA in control Tcells is 100%. Data are pooled from two independent experiments. D, Sample Western blot analysis of mitochondrial proteins in total thymocytes and sortedperipheral T cells from Atg7f/f and Atg7f/fLck-Cre T cells. Values listed below blot were normalized to actin, and wild-type CD4� T cells were arbitrarilyset to 100. E, Quantification of intensity of Western blots from multiple experiments. All values were normalized to actin, and CD4� T cells were arbitrarilygiven the value of 100 in each experiment. Tom20 and CytC include three to six mice from three independent experiments; AIF and mtHsp70 include twoto four mice from two independent experiments.

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discrepancy is unknown but may reflect subtle differences in thetwo model systems or the activity of Atg5 and Atg7 in T cellautophagy.

When MitoTracker staining values from multiple mice werenormalized to DP cell mean fluorescence, we observed a consistent50–75% reduction in the staining of wild-type T lymphocytes asthey transitioned from the thymus to peripheral circulation (Fig.4D). This reduction was larger in CD8� than CD4� T cells, whichmay reflect the intermediate mitochondrial content phenotype inCD8� SP thymocytes (Fig. 4D). In contrast, mitochondrial contentwas not changed in developing B lymphocytes from CD43�

B220� B cell precursors to peripheral mature B cell populations(Fig. 4E). When Atg7f/fLck-Cre MitoTracker Green values werenormalized to control DP fluorescence, it became clear that therelative MFI increase in autophagy-deficient peripheral T lympho-cytes largely reflects an impaired reduction in mitochondrial con-tent when compared with thymocytes (Fig. 4D). Thus, autophagy-dependent regulated changes in mitochondrial content mark theimportant developmental transition from thymocyte to circulatingperipheral T lymphocyte.

Mitochondrial morphology and DNA content in Atg7-deficientT cells

Given the dramatic changes in mitochondrial content, particu-larly in CD8� Atg7f/fLck-Cre peripheral T cells, we sorted au-tophagy-deficient and control CD8� T cells to visually exam-ined mitochondria by transmission electron microscopy.Although there was notable variation in mitochondrial morphol-ogy between individual cell cross-sections, the mitochondrialshape and appearance in Atg7f/fLck-Cre T cells were largelycomparable to controls (Fig. 5A). The mitochondria in autoph-

agy-deficient T cells exhibited clear outer membranes and cris-tae without specific evidence of dysfunction, including swellingor condensation (Fig. 5A).

To determine whether differences observed in mitochondrialcontent from MitoTracker Green staining might reflect differ-ences in mitochondrial volume in these cells, we calculatedmitochondrial surface area in �40 cell cross-sections by man-ually outlining mitochondria using a quantification tool in Axio-Vision (Zeiss). Large variations between individual cross-sec-tions were found in all samples analyzed (Fig. 5B) and likelyreflect the uneven distribution of mitochondria in three-dimen-sional space within the cytoplasm of T cells observed by im-munofluorescent microscopy. Nevertheless, a statistically sig-nificant increase in mitochondrial surface in Atg7-deficientCD8� T cells was observed when compared with control T cells(Fig. 5B). To confirm this finding, we used a third measure ofmitochondrial content in cells, mitochondrial DNA copy num-ber (40). By quantitative PCR analysis, we found that sortedAtg7f/fLck-Cre CD8�CD44low T cells contained �2-fold moremitochondrial DNA than did control cells (Fig. 5C). Finally, weperformed Western blots for a range of mitochondrial proteinsin Atg7f/f and Atg7f/fLck-Cre thymocytes and sorted T cells.These results demonstrated a large increase in the inner-mem-brane and intermembrane space proteins cytochrome C andAIF, with less dramatic changes in the outer mitochondrialtransport protein Tom20 and inner matrix chaperone mtHsp70(Fig. 5, D and E). Taken together, these results suggest that inthe absence of autophagy increased numbers of grossly normalmitochondria are present in mature T cells and that autophagyis critical to clear mitochondria in these cells.

FIGURE 6. Increased ROS production and imbalanced expression of pro- and antiapoptotic proteins in Atg7-deficient T cells. A, ROS production incontrol and Atg7-deficient CD4� T cells cultured overnight in 1 ng/ml IL-7. ROS was measured by incubating cells for 1 h at 37°C with 5 �M eitherdihydroethidium (DHE) or CM-H2DCFDA (DCF) and measuring mean cell fluorescence by flow cytometry. Data depicted are from two independentexperiments for each detection reagent. B and D, Flow cytometry analysis of intracellular Bcl-2 and Mcl-1 expression in Atg7f/f (dark shaded histogram)and Atg7f/fLck-Cre (black line open histogram) CD44low naive T cells. Isotype controls for Atg7f/f (light shaded histogram) and Atg7f/fLck-Cre (gray lineopen histogram) cells are depicted and numeric values represent an index of Bcl-2 or Mcl-1 expression divided by isotype MFI. Values from two to threeindependent experiments are pooled in graphical form as a percentage of Atg7-deficient over control indexed fluorescence. C and E, Western blot of sortedCD4� and CD8� T cells from Atg7f/f and Atg7f/fLck-Cre mice (�98% pure) for Bcl-2 and Mcl-1 expression. Data are representative of two independentexperiments. F, Western blot of enriched Atg7-deficient and control spleen and lymph node T cells (�95% pure) for Bax and Bak. Numbers represent bandintensity normalized to actin loading controls. Data are representative of two independent experiments.

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Increased ROS production and imbalanced expression ofpro- and antiapoptotic proteins in Atg7-deficient T lymphocytes

Mitochondria are responsible for numerous important metabolicpathways within eukaryotic cells. To determine whether increasedmitochondrial content in autophagy-deficient T cells was associ-ated with functional changes in these cells, we examined ROSproduction in cultured T lymphocytes. We observed a 2-fold in-crease in ROS production by Atg7-deficient T cells as measured bydihydroethidium and CM-H2DCFDA staining (Fig. 6A). ROS pro-duction has been shown to play a proapoptotic role in T cell hy-bridomas as well as in activated primary T cells (54, 55).

In addition to producing ROS, mitochondria can become toxicto cells through their role in the intrinsic death pathway. Deathinitiated through the mitochondria is largely regulated by Bcl-2family proteins and classically results in the release of proapop-totic factors normally sequestered away from downstream effectorsin the mitochondrial intermembrane space (48). To determinewhether abnormalities in Bcl-2 family protein expression are as-sociated with enhanced apoptosis in autophagy-deficient mature Tlymphocytes, we first examined the expression of the two majorantiapoptotic Bcl-2 family members expressed in naive T cells,Bcl-2 and Mcl-1 (56, 57). The expression of Bcl-2 as assessed byboth flow cytometry and Western blot was increased in Atg7f/fLck-Cre T cells (Fig. 6, B and C). Conversely, we found no significantincrease in Mcl-1 expression in Atg7f/fLck-Cre cells as measuredby both flow cytometry and Western blot (Fig. 6, D and E). Weobserved low levels of Bcl-xL expression as assessed by Westernblot in both Atg7-deficient and control T cells, consistent with thelow expression of this antiapoptotic protein in naive T lympho-cytes (unpublished data). Thus, T cells lacking the essential auto-phagy gene Atg7 demonstrate a selective up-regulation of Bcl-2,but not other antiapoptotic family members.

We next examined the proapoptotic members of the Bcl-2 family,Bax and Bak. While Bax protein levels remained unchanged in au-tophagy-deficient T lymphocytes, Bak protein was increased �2-foldby Western blot analysis (Fig. 6F). Additionally, consistent with theincrease in mitochondrial content in our autophagy-deficient T cells,we found a significant increase in death-inducing mitochondrial pro-teins cytochrome c and AIF expression in Atg7f/fLck-Cre T cellswhen compared with Atg7f/f controls (Fig. 5, D and E). Given thecentral role of the BH3 only protein Bim in the induction of naive andactivated T cell death (58), we also examined Bim expression butobserved no difference between autophagy-deficient and control Tcells (unpublished data). Taken together, these data suggest thatperturbations in the balance of pro- and antiapoptotic proteins maycontribute to cell death in primary T cells in the absence of autophagy.

DiscussionAlthough autophagy has long been recognized, its functions invarious physiological and pathological processes have only begunto be elucidated. Our studies have revealed several key findingsregarding the role of autophagy in regulating T lymphocyte sur-vival. First, our data strongly suggest that autophagy itself is es-sential for mature T cell survival. Although our previous resultsshow that Atg5-deficient mature T cells undergo massive apoptosis(35), it was not clear whether the defective survival of Atg5-defi-cient T cells was caused by the role of Atg5 in the induction ofautophagy or other survival pathways, as Atg5 interacts withFADD and Bcl-xL (44, 45). The fact that T lymphocytes lacking asecond essential autophagy gene, Atg7, exhibit a similar survivaldefect to that of Atg5-deficient T cells strongly argues for a role forautophagy in maintaining mature T cell survival. Second, we showthat mitochondrial content in T lymphocytes is developmentally

regulated. A reduction of mitochondrial content marks the transi-tion from thymocytes to peripheral mature T cells. Third, the clear-ance of superfluous mitochondria in mature T cells critically de-pends on autophagy. This is supported by our measurement ofmitochondrial content through FACS analysis of MitoTrackerGreen-stained mitochondria, direct survey of mitochondrial sur-face area under transmission electron microscopy, quantitativedetermination of mitochondrial DNA, as well as the expressionlevels of various proteins associated with mitochondria. Interest-ingly, this phenomenon appears to be limited to T lymphocytes, asmitochondrial content is not developmentally regulated in B lym-phocytes. Fourth, in the absence of autophagy, we have observednot only enhanced mitochondrial content but also abnormality inmitochondria-associated functions, including ROS production andapoptosis.

The precise role of autophagy in cell survival and cell deathremains controversial, and this controversy likely reflects the com-plex functional and molecular intersection of autophagy with pro-survival and pro-death pathways (46, 59, 60). In cells where apo-ptosis is inhibited, the degradation of cytoplasmic material inautophagosomes protects cells from death by providing essentialmetabolic support during periods of nutrient deprivation (9); how-ever, the autophagic degradation of essential proteins, includingcatalase (61, 62), and organelles (20) can also commit theses cellto death. Although the outcome of autophagosome formation incell survival may depend on cell type and context, autophagyclearly serves a critical role in the homeostatic survival of cells invivo. This has been most clearly demonstrated in the CNS, wherethe conditional deletion of the essential autophagy genes Atg5 orAtg7 leads to neurodegeneration associated with an increase inTUNEL-positive apoptotic cells (10, 11). Our results here, togetherwith our previous data (35), demonstrate that deletion of Atg5 orAtg7 in primary mouse T lymphocytes leads to a dramatic impair-ment of naive T cell peripheral survival with up to a 75% reductionin the number of circulating T cells. Thus autophagy constitutes anovel pro-survival pathway in mature T lymphocytes.

Our finding that mitochondrial content is developmentallyregulated in T lymphocytes and the reduction of mitochondrialcontent in peripheral T cells critically depends on autophagyhas identified a novel mechanism by which mature T lympho-cyte homeostasis is maintained. Thymocytes from DN, DP, andCD4� SP compartments contain high content of mitochondria.In contrast, mitochondrial content in CD8� SP thymocytes isreduced to an intermediate level. Furthermore, although mito-chondrial content in CD4� mature T cells is reduced by �50%when compared with DP thymocytes, this reduction is �80%for CD8� mature T cells. This more dramatic reduction of mi-tochondrial content in developing CD8� T cells may reflecttheir lower tolerance for superfluous mitochondria than inCD4� mature T lymphocytes. Consistent with this notion,CD8� T cells lacking Atg5 (35) or Atg7 exhibit higher apo-ptosis rates and more dramatic reductions in cell numbers thando autophagy-deficient CD4� T cells. Interestingly, althoughmitochondrial content is increased, morphology appears normalunder transmission electron microscopy. This is in contrast tothe deformed mitochondria in Atg7-deficient hepatocytes (8).This difference may suggest a role of autophagy in removingsuperfluous mitochondria in T cells and damaged mitochondriain liver cells. Alternatively, low levels of mitochondrial damagemay result in the rapid apoptosis of T lymphocytes, makingabnormal mitochondria more difficult to detect among live au-tophagy-deficient T cells.

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In addition to T lymphocytes, two other cell types appear toclear mitochondria and other intracellular organelles during devel-opment. It has been observed that intracellular organelles in epi-thelial cells during lens development and in erythrocytes duringerythroid cell maturation are rapidly eliminated, and autophagy hasbeen proposed as a mechanism for the degradation of these or-ganelles (14, 21, 22, 24). However, recent data using Atg5-defi-cient mice have demonstrated that organelle degradation duringlens and erythroid differentiation is independent of this essentialautophagy gene (23). Therefore, T lymphocytes provide an impor-tant example of cells that undergo autophagy-dependent organelleclearance in vivo.

Why does mitochondrial content need to be down-regulated asT lymphocytes undergo maturation from thymocytes to mature Tcells? As the transition from thymocytes to mature T cells marksa dramatic change in their environment, factors in the periphery(blood and secondary lymphoid organs) may cause stress to matureT cells if mitochondrial content remains static. One such likelyenvironmental factor is the oxygen level in the periphery. It hasbeen shown that the oxygen tension in blood (5–13 kPa) (63) isdramatically higher than in the thymus (1.3 kPa) (64). High mito-chondrial content in circulating mature T cells may lead to theproduction of excess amount of ROS, a byproduct of mitochon-drial respiration. As ROS is toxic to T lymphocytes (54, 55), clear-ance of superfluous mitochondria by autophagy in mature T cellsmay be necessary to avoid ROS toxicity. Consistent with this no-tion, we observed higher ROS production in autophagy-deficient Tcells. This idea is further supported by recent evidence showingthat ROS induces autophagy through Atg4 (65). Thus, peripheralmature T cells may use a physiologically relevant stress, high ox-ygen tension and ROS, to induce autophagy to remove extramitochondria.

The apoptosis of autophagy-deficient T cells is likely caused bymultiple factors. While enhanced ROS may cause T cell death, theenhanced expression of proapoptotic Bak may further contribute toapoptosis of autophagy-deficient T cells. Although Bcl-2 expres-sion is increased in autophagy-deficient T cells, this level of Bcl-2may not be sufficient to prevent Bak-dependent apoptosis. A pre-vious report has shown that Bak can be effectively sequestered byMcl-1 and Bcl-xL but not by Bcl-2 (66). Although Mcl-1 is alsoassociated with mitochondrial membrane, its expression level inautophagy-deficient T cells is not obviously changed. The reasonfor this observation is not clear, but may be related to the ex-tremely short half-life of Mcl-1 in cells as well as the tightregulation of this protein’s stability by upstream signaling path-ways within the cell (67, 68). Additionally, the high expressionlevel of cytochrome c in autophagy-deficient T cells may fur-ther prime these cells to caspase activation and cell death. Thus,it is likely that autophagy-deficient T cells die due to the acti-vation of caspases by multiple factors derived from the super-fluous mitochondria. Interestingly, increased mitochondrialcontent in HIV-specific CD8� T cells in human patients alsocorrelates with enhanced susceptibility of these cells to apopto-sis, suggesting that mitochondrial homeostasis may play an im-portant role in T cell survival in normal physiology as well asin pathologic situations (51).

AcknowledgmentsWe thank Dr. Jeffrey Rathmell for useful discussion and critical review ofthis manuscript, Dr. Tso-Pang Yao for discussion and sharing of reagents,Dr. Sara Miller and Philip Christopher for help in transmission electronmicroscopy, and Michael Forrester for ROS assays.

DisclosuresThe authors have no financial conflicts of interest.

References1. Klionsky, D. J., and S. D. Emr. 2000. Autophagy as a regulated pathway of

cellular degradation. Science 290: 1717–1721.2. Levine, B., and G. Kroemer. 2008. Autophagy in the pathogenesis of disease.

Cell 132: 27–42.3. Mortimore, G. E., and A. R. Poso. 1987. Intracellular protein catabolism and its

control during nutrient deprivation and supply. Annu. Rev. Nutr. 7: 539–564.4. Mizushima, N., Y. Ohsumi, and T. Yoshimori. 2002. Autophagosome formation

in mammalian cells. Cell Struct. Funct. 27: 421–429.5. Lum, J. J., R. J. DeBerardinis, and C. B. Thompson. 2005. Autophagy in meta-

zoans: cell survival in the land of plenty. Nat. Rev. Mol. Cell Biol. 6: 439–448.6. Kim, I., S. Rodriguez-Enriquez, and J. J. Lemasters. 2007. Selective degradation

of mitochondria by mitophagy. Arch. Biochem. Biophys. 462: 245–253.7. Kuma, A., M. Hatano, M. Matsui, A. Yamamoto, H. Nakaya, T. Yoshimori,

Y. Ohsumi, T. Tokuhisa, and N. Mizushima. 2004. The role of autophagy duringthe early neonatal starvation period. Nature 432: 1032–1036.

8. Komatsu, M., S. Waguri, T. Ueno, J. Iwata, S. Murata, I. Tanida, J. Ezaki,N. Mizushima, Y. Ohsumi, Y. Uchiyama, et al. 2005. Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice. J. Cell Biol. 169:425–434.

9. Lum, J. J., D. E. Bauer, M. Kong, M. H. Harris, C. Li, T. Lindsten, andC. B. Thompson. 2005. Growth factor regulation of autophagy and cell survivalin the absence of apoptosis. Cell 120: 237–248.

10. Komatsu, M., S. Waguri, T. Chiba, S. Murata, J. Iwata, I. Tanida, T. Ueno,M. Koike, Y. Uchiyama, E. Kominami, and K. Tanaka. 2006. Loss of autophagyin the central nervous system causes neurodegeneration in mice. Nature 441:880–884.

11. Hara, T., K. Nakamura, M. Matsui, A. Yamamoto, Y. Nakahara,R. Suzuki-Migishima, M. Yokoyama, K. Mishima, I. Saito, H. Okano, andN. Mizushima. 2006. Suppression of basal autophagy in neural cells causes neu-rodegenerative disease in mice. Nature 441: 885–889.

12. Kundu, M., and C. B. Thompson. 2005. Macroautophagy versus mitochondrialautophagy: a question of fate? Cell Death Differ. 12(Suppl. 2): 1484–1489.

13. Mortimore, G. E., G. Miotto, R. Venerando, and M. Kadowaki. 1996. Autophagy.Subcell. Biochem. 27: 93–135.

14. Takano-Ohmuro, H., M. Mukaida, E. Kominami, and K. Morioka. 2000. Auto-phagy in embryonic erythroid cells: its role in maturation. Eur. J. Cell Biol. 79:759–764.

15. Takeshige, K., M. Baba, S. Tsuboi, T. Noda, and Y. Ohsumi. 1992. Autophagyin yeast demonstrated with proteinase-deficient mutants and conditions for itsinduction. J. Cell Biol. 119: 301–311.

16. Ravikumar, B., Z. Berger, C. Vacher, C. J. O’Kane, and D. C. Rubinsztein. 2006.Rapamycin pre-treatment protects against apoptosis. Hum. Mol. Genet. 15:1209–1216.

17. Colell, A., J. E. Ricci, S. Tait, S. Milasta, U. Maurer, L. Bouchier-Hayes,P. Fitzgerald, A. Guio-Carrion, N. J. Waterhouse, C. W. Li, et al. 2007. GAPDHand autophagy preserve survival after apoptotic cytochrome c release in the ab-sence of caspase activation. Cell 129: 983–997.

18. Elmore, S. P., T. Qian, S. F. Grissom, and J. J. Lemasters. 2001. The mitochon-drial permeability transition initiates autophagy in rat hepatocytes. FASEB J. 15:2286–2287.

19. Rodriguez-Enriquez, S., I. Kim, R. T. Currin, and J. J. Lemasters. 2006. Trackerdyes to probe mitochondrial autophagy (mitophagy) in rat hepatocytes. Autoph-agy 2: 39–46.

20. Xue, L., G. C. Fletcher, and A. M. Tolkovsky. 2001. Mitochondria are selectivelyeliminated from eukaryotic cells after blockade of caspases during apoptosis.Curr. Biol. 11: 361–365.

21. Bassnett, S. 2002. Lens organelle degradation. Exp. Eye Res. 74: 1–6.22. Kent, G., O. T. Minick, F. I. Volini, and E. Orfei. 1966. Autophagic vacuoles in

human red cells. Am. J. Pathol. 48: 831–857.23. Matsui, M., A. Yamamoto, A. Kuma, Y. Ohsumi, and N. Mizushima. 2006.

Organelle degradation during the lens and erythroid differentiation is independentof autophagy. Biochem. Biophys. Res. Commun. 339: 485–489.

24. Sandoval, H., P. Thiagarajan, S. K. Dasgupta, A. Schumacher, J. T. Prchal,M. Chen, and J. Wang. 2008. Essential role for Nix in autophagic maturation oferythroid cells. Nature 454: 232–235.

25. Schmid, D., and C. Munz. 2007. Innate and adaptive immunity through autoph-agy. Immunity 27: 11–21.

26. Deretic, V. 2006. Autophagy as an immune defense mechanism. Curr. Opin.Immunol. 18: 375–382.

27. Nakagawa, I., A. Amano, N. Mizushima, A. Yamamoto, H. Yamaguchi,T. Kamimoto, A. Nara, J. Funao, M. Nakata, K. Tsuda, et al. 2004. Autophagydefends cells against invading group A Streptococcus. Science 306: 1037–1040.

28. Gutierrez, M. G., S. S. Master, S. B. Singh, G. A. Taylor, M. I. Colombo, andV. Deretic. 2004. Autophagy is a defense mechanism inhibiting BCG and My-cobacterium tuberculosis survival in infected macrophages. Cell 119: 753–766.

29. Singh, S. B., A. S. Davis, G. A. Taylor, and V. Deretic. 2006. Human IRGMinduces autophagy to eliminate intracellular mycobacteria. Science 313:1438–1441.

30. Andrade, R. M., M. Wessendarp, M. J. Gubbels, B. Striepen, and C. S. Subauste.2006. CD40 induces macrophage anti-Toxoplasma gondii activity by triggeringautophagy-dependent fusion of pathogen-containing vacuoles and lysosomes.J. Clin. Invest. 116: 2366–2377.

4054 AUTOPHAGY IN T CELL MITOCHONDRIA HOMEOSTASIS

by guest on April 12, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

31. Ling, Y. M., M. H. Shaw, C. Ayala, I. Coppens, G. A. Taylor, D. J. Ferguson, andG. S. Yap. 2006. Vacuolar and plasma membrane stripping and autophagic elim-ination of Toxoplasma gondii in primed effector macrophages. J. Exp. Med. 203:2063–2071.

32. Schmid, D., M. Pypaert, and C. Munz. 2007. Antigen-loading compartments formajor histocompatibility complex class II molecules continuously receive inputfrom autophagosomes. Immunity 26: 79–92.

33. Paludan, C., D. Schmid, M. Landthaler, M. Vockerodt, D. Kube, T. Tuschl, andC. Munz. 2005. Endogenous MHC class II processing of a viral nuclear antigenafter autophagy. Science 307: 593–596.

34. Gerland, L. M., L. Genestier, S. Peyrol, M. C. Michallet, S. Hayette,I. Urbanowicz, P. Ffrench, J. P. Magaud, and M. Ffrench. 2004. Autolysosomesaccumulate during in vitro CD8� T-lymphocyte aging and may participate ininduced death sensitization of senescent cells. Exp. Gerontol. 39: 789–800.

35. Pua, H. H., I. Dzhagalov, M. Chuck, N. Mizushima, and Y. W. He. 2007. Acritical role for the autophagy gene Atg5 in T cell survival and proliferation.J. Exp. Med. 204: 25–31.

36. Espert, L., M. Denizot, M. Grimaldi, V. Robert-Hebmann, B. Gay, M. Varbanov,P. Codogno, and M. Biard-Piechaczyk. 2006. Autophagy is involved in T celldeath after binding of HIV-1 envelope proteins to CXCR4. J. Clin. Invest. 116:2161–2172.

37. Li, C., E. Capan, Y. Zhao, J. Zhao, D. Stolz, S. C. Watkins, S. Jin, and B. Lu.2006. Autophagy is induced in CD4� T cells and important for the growth factor-withdrawal cell death. J. Immunol. 177: 5163–5168.

38. Miller, B. C., Z. Zhao, L. M. Stephenson, K. Cadwell, H. H. Pua, H. K. Lee,N. N. Mizushima, A. Iwasaki, Y. W. He, W. Swat, and H. W. Virgin. 2007. Theautophagy gene ATG5 plays an essential role in B lymphocyte development.Autophagy 4: 309–314.

39. Hennet, T., F. K. Hagen, L. A. Tabak, and J. D. Marth. 1995. T-cell-specificdeletion of a polypeptide N-acetylgalactosaminyl-transferase gene by site-di-rected recombination. Proc. Natl. Acad. Sci. USA 92: 12070–12074.

40. Masuyama, M., R. Iida, H. Takatsuka, T. Yasuda, and T. Matsuki. 2005. Quan-titative change in mitochondrial DNA content in various mouse tissues duringaging. Biochim. Biophys. Acta 1723: 302–308.

41. Hance, N., M. I. Ekstrand, and A. Trifunovic. 2005. Mitochondrial DNA poly-merase gamma is essential for mammalian embryogenesis. Hum. Mol. Genet. 14:1775–1783.

42. Pfaffl, M. W. 2001. A new mathematical model for relative quantification inreal-time RT-PCR. Nucleic Acids Res. 29: e45.

43. Pfaffl, M. W., G. W. Horgan, and L. Dempfle. 2002. Relative expression softwaretool (REST) for group-wise comparison and statistical analysis of relative ex-pression results in real-time PCR. Nucleic Acids Res. 30: e36.

44. Pyo, J. O., M. H. Jang, Y. K. Kwon, H. J. Lee, J. I. Jun, H. N. Woo, D. H. Cho,B. Choi, H. Lee, J. H. Kim, et al. 2005. Essential roles of Atg5 and FADD inautophagic cell death: dissection of autophagic cell death into vacuole formationand cell death. J. Biol. Chem. 280: 20722–20729.

45. Yousefi, S., R. Perozzo, I. Schmid, A. Ziemiecki, T. Schaffner, L. Scapozza,T. Brunner, and H. U. Simon. 2006. Calpain-mediated cleavage of Atg5 switchesautophagy to apoptosis. Nat. Cell Biol. 8: 1124–1132.

46. Maiuri, M. C., E. Zalckvar, A. Kimchi, and G. Kroemer. 2007. Self-eating andself-killing: crosstalk between autophagy and apoptosis. Nat. Rev. Mol. Cell Biol.8: 741–752.

47. Zhang, N., H. Hartig, I. Dzhagalov, D. Draper, and Y. W. He. 2005. The role ofapoptosis in the development and function of T lymphocytes. Cell Res. 15:749–769.

48. Rathmell, J. C., and C. B. Thompson. 1999. The central effectors of cell death inthe immune system. Annu. Rev. Immunol. 17: 781–828.

49. Pendergrass, W., N. Wolf, and M. Poot. 2004. Efficacy of MitoTracker Green andCMXrosamine to measure changes in mitochondrial membrane potentials in liv-ing cells and tissues. Cytometry A 61: 162–169.

50. Presley, A. D., K. M. Fuller, and E. A. Arriaga. 2003. MitoTracker Green label-ing of mitochondrial proteins and their subsequent analysis by capillary electro-phoresis with laser-induced fluorescence detection. J. Chromatogr. B Analyt.Technol. Biomed. Life Sci. 793: 141–150.

51. Petrovas, C., Y. M. Mueller, I. D. Dimitriou, S. R. Altork, A. Banerjee, P. Sklar,K. C. Mounzer, J. D. Altman, and P. D. Katsikis. 2007. Increased mitochondrialmass characterizes the survival defect of HIV-specific CD8� T cells. Blood 109:2505–2513.

52. Nagy, G., M. Barcza, N. Gonchoroff, P. E. Phillips, and A. Perl. 2004. Nitricoxide-dependent mitochondrial biogenesis generates Ca2� signaling profile oflupus T cells. J. Immunol. 173: 3676–3683.

53. Schweers, R. L., J. Zhang, M. S. Randall, M. R. Loyd, W. Li, F. C. Dorsey,M. Kundu, J. T. Opferman, J. L. Cleveland, J. L. Miller, and P. A. Ney. 2007.NIX is required for programmed mitochondrial clearance during reticulocytematuration. Proc. Natl. Acad. Sci. USA 104: 19500–19505.

54. Sandstrom, P. A., and T. M. Buttke. 1993. Autocrine production of extracellularcatalase prevents apoptosis of the human CEM T-cell line in serum-free medium.Proc. Natl. Acad. Sci. USA 90: 4708–4712.

55. Hildeman, D. A., T. Mitchell, T. K. Teague, P. Henson, B. J. Day, J. Kappler, andP. C. Marrack. 1999. Reactive oxygen species regulate activation-induced T cellapoptosis. Immunity 10: 735–744.

56. Veis, D. J., C. L. Sentman, E. A. Bach, and S. J. Korsmeyer. 1993. Expressionof the Bcl-2 protein in murine and human thymocytes and in peripheral T lym-phocytes. J. Immunol. 151: 2546–2554.

57. Opferman, J. T., A. Letai, C. Beard, M. D. Sorcinelli, C. C. Ong, andS. J. Korsmeyer. 2003. Development and maintenance of B and T lymphocytesrequires antiapoptotic MCL-1. Nature 426: 671–676.

58. Marrack, P., and J. Kappler. 2004. Control of T cell viability. Annu. Rev. Immu-nol. 22: 765–787.

59. Debnath, J., E. H. Baehrecke, and G. Kroemer. 2005. Does autophagy contributeto cell death? Autophagy 1: 66–74.

60. Maiuri, M. C., G. Le Toumelin, A. Criollo, J. C. Rain, F. Gautier, P. Juin,E. Tasdemir, G. Pierron, K. Troulinaki, N. Tavernarakis, et al. 2007. Functionaland physical interaction between Bcl-XL and a BH3-like domain in Beclin-1.EMBO J. 26: 2527–2539.

61. Yu, L., A. Alva, H. Su, P. Dutt, E. Freundt, S. Welsh, E. H. Baehrecke, andM. J. Lenardo. 2004. Regulation of an ATG7-beclin 1 program of autophagic celldeath by caspase-8. Science 304: 1500–1502.

62. Yu, L., F. Wan, S. Dutta, S. Welsh, Z. Liu, E. Freundt, E. H. Baehrecke, andM. Lenardo. 2006. Autophagic programmed cell death by selective catalase deg-radation. Proc. Natl. Acad. Sci. USA 103: 4952–4957.

63. Sitkovsky, M., and D. Lukashev. 2005. Regulation of immune cells by local-tissue oxygen tension: HIF1� and adenosine receptors. Nat. Rev. Immunol. 5:712–721.

64. Braun, R. D., J. L. Lanzen, S. A. Snyder, and M. W. Dewhirst. 2001. Comparisonof tumor and normal tissue oxygen tension measurements using OxyLite or mi-croelectrodes in rodents. Am. J. Physiol. 280: H2533–H2544.

65. Scherz-Shouval, R., E. Shvets, E. Fass, H. Shorer, L. Gil, and Z. Elazar. 2007.Reactive oxygen species are essential for autophagy and specifically regulate theactivity of Atg4. EMBO J. 26: 1749–1760.

66. Willis, S. N., L. Chen, G. Dewson, A. Wei, E. Naik, J. I. Fletcher, J. M. Adams,and D. C. Huang. 2005. Proapoptotic Bak is sequestered by Mcl-1 and Bcl-xL, butnot Bcl-2, until displaced by BH3-only proteins. Genes Dev. 19: 1294–1305.

67. Michels, J., P. W. Johnson, and G. Packham. 2005. Mcl-1. Int. J. Biochem. CellBiol. 37: 267–271.

68. Maurer, U., C. Charvet, A. S. Wagman, E. Dejardin, and D. R. Green. 2006.Glycogen synthase kinase-3 regulates mitochondrial outer membrane permeabi-lization and apoptosis by destabilization of MCL-1. Mol. Cell. 21: 749–760.

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