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Is Essential for Mitochondrial Clearance in Mature T Lymphocytes Heather H. Pua, Jian Guo, Masaaki Komatsu and You-Wen He This information is current as of September 28, 2021. J Immunol 2009; 182:4046-4055; ; doi: 10.4049/jimmunol.0801143 http://www.jimmunol.org/content/182/7/4046 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Autophagy Is Essential for Mitochondrial Clearance in Mature T Lymphocytes1

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

Macroautophagy plays an important role in the regulation of survival, metabolism, and the lysosomal degradation of cytoplasmic material. In the immune system, autophagy contributes to the clearance of intracellular pathogens, MHCII cross-presentation of endogenous Ags, as well as cell survival. We and others have demonstrated that autophagy occurs in T lymphocytes and contributes to the regulation of their cellular function, including survival and proliferation. Here we show that 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 thymus marking 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 , and autophagy- Downloaded from deficient mature T cells fail to reduce their mitochondrial content in vivo. Consistent with alterations in mitochondrial content, autophagy-deficient T cells have increased production as well as an imbalance in pro- and antiapoptotic expression. With much recent interest in the possibility of autophagy-dependent developmentally programmed clearance of organelles in lens epithelial cells and erythrocytes, our data demonstrate that autophagy may have a physiologically significant role in the clearance of superfluous mitochondria in T lymphocytes as part of normal T cell

. The Journal of Immunology, 2009, 182: 4046–4055. http://www.jimmunol.org/

acroautophagy (hereafter referred to as autophagy) is starvation or deprivation. This is best demonstrated a well-conserved catabolic process in eukaryotic in mice lacking Atg5 or Atg7, which both succumb to starvation as M cells characterized by the formation of double- neonates before robust suckling (7, 8). Importantly, it has also been membrane vesicles 0.5–1.5 ␮m in diameter in the of shown that the survival of an IL-3-dependent hematopoietic cell cells termed (1, 2). Autophagosomes arise line during deprivation depends on for- through the elongation of cup-shaped isolation membranes that mation to provide metabolic substrates (9). Additionally, autoph- form spherical vesicles before fusing with to become agy-mediated clearance of toxic cytoplasmic materials is critical degradative compartments. Mature autophagosomes encase both for neural cell survival, as conditional deletion of Atg5 or Atg7 in by guest on September 28, 2021 as well as organelles, consistent with the early character- neurons leads to the accumulation of -positive inclusions ization of autophagy as a major pathway for protein degradation and widespread neural cell (10, 11). during periods of starvation (3). Autophagy induction is regulated Mounting evidence implicates autophagy in mitochondrial by the activity of class III PI3K in complex with the essential removal (often designated “”) in both mammalian autophagy gene Beclin-1 ( Atg6). Two ubiquitin-like conju- and yeast cells (12). Mitochondria have been found within ves- gation pathways involving the autophagy genes Atg3, Atg5, Atg7, icles that possess double or multiple membranes in rat hepato- -associated protein light chain 3 (LC3, yeast Atg8), cytes (13), hamster erythroid cells (14), and yeast (15). Mod- Atg10, and Atg12 are also required for the formation of autopha- ulating autophagy by rapamycin treatment or expression of gosomes (1, 4). GAPDH affects mitochondrial mass within cells (16, 17). Mi- The induction of autophagy generally executes two complemen- tochondrial permeability transition triggers autophagy-medi- tary functions in eukaryotes that include the recycling of useful ated mitochondrial degradation (18, 19). Mitophagy is also in- metabolic substrates as well as the removal of cytoplasmic mate- duced by cytotoxic agents in the presence of caspase inhibitors rial (1, 5, 6). The digestion of intracellular to generate (20). In vivo, there has been much interest in the physiologic metabolic substrates is essential for cell survival during periods of role of autophagy in mitochondrial clearance during lens and erythroid differentiation (14, 21–24). Within immunology, there is an expanding role for autophagy in *Department of Immunology, Duke University Medical Center, Durham, NC 27710; both the innate and adaptive immune system (25, 26). Autophagy and †PRESTO (Precursory Research for Embryonic Science and Technology), Japan contributes to the clearance of intracellular pathogens (27–31) as Science and Technology Corporation, Kawaguchi and Laboratory of Frontier Science, Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo, Japan well as the MHCII cross-presentation of endogenous Ags (32, 33). Received for publication April 8, 2008. Accepted for publication January 26, 2009. We and others have also demonstrated a role for autophagy in lymphocytes. In T lymphocytes, double-membrane autophago- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance somes form in both human and murine T cells and can be induced with 18 U.S.C. Section 1734 solely to indicate this fact. in TCR-stimulated proliferating cells in vitro (34–37). Although 1 This work was supported by National Institutes of Health Grant AI-073947. the functional consequences of autophagosome formation in lym- 2 Address correspondence and reprint requests to Dr. You-Wen He, Department of phocytes are not well understood, genetic and molecular studies Immunology, Duke University Medical Center, Box 3010, Durham, NC 27710. have demonstrated a complex role for autophagy in T cell survival E-mail address: [email protected] and function. Loss of the essential autophagy gene Atg5 impairs Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 the survival and proliferation of mature T cells in vivo (35), while www.jimmunol.org/cgi/doi/10.4049/jimmunol.0801143 The Journal of Immunology 4047 induction of autophagy by the HIV envelope glycoprotein induces incubated with 0.5% uranyl acetate in veronal acetate buffer for1hatroom death in target lymphocytes (36). In B lymphocytes, autophagy is temperature. Specimens were then dehydrated in an ascending series of required for survival of developing pre-B cells as well as mature ethanol (35%, 70%, 95%, and two changes of 100%) for 10 min each, followed by two changes of propylene oxide for 5 min each. The samples B1 B cells (38). were incubated with a 1:1 mixture of 100% resin and propylene oxide for In this paper, we demonstrate that the essential autophagy gene 1 h, followed by two changes of 100% resin, each for 30 min. Finally, the Atg7 is required for mature T cell survival. We show that mito- samples were embedded in resin and polymerized at 60°C overnight. Thick chondrial content is developmentally regulated in T lymphocytes sections (0.5 ␮m) were cut and stained with toluidine blue for light mi- croscopy selection of the appropriate area for ultrathin sections. Thin sec- and that autophagy is critical for the clearance of mitochondrial tions (60–90 nm) were cut, mounted on copper grids, and poststained with content in mature T cells. uranyl acetate and lead citrate. Micrographs were taken with a Philips LS 410 electron microscope. Images were analyzed using AxioVision software Materials and Methods (Zeiss). Animals Reactive oxygen species (ROS) assay Atg7-floxed and Atg5-deficient mice were generated and characterized pre- ␮ viously (7, 8). To generate mice with Atg7-deficient T lymphocytes, Atg7- To assay for ROS production, lymphocytes were incubated in 5 M di- floxed mice were crossed to Lck-Cre transgenic mice (39) (The Jackson hydroethidium (Sigma-Aldrich) or CM-H2DCFDA (Molecular Probes) for Laboratory). Atg7 genomic deletion was assessed by PCR detection of the 1 h in RPMI 1640 complete medium at 37°C and subsequently analyzed by flow cytometry. ROS production was inhibited in vitro by the addition of Atg7-floxed allele (forward, TGA GAC ATG GCC TGA AGA AAC CCA; ␮ reverse, ATG CTG CAG GAC AGA GAC CAT CA) and the wild-type 150 M manganese (III) tetrakis 4-benzoic acid (MnTBAP; Calbiochem), genomic locus (forward, TTA CAG TCG GCC AGG CTG AC; reverse, 1mMN-acetylcysteine (Sigma-Aldrich), 50 U/ml superoxide dismutase- CCT GGG CTG CCA GAA TTT CTC) in FACS-sorted thymocytes and polyethylene glycol (Sigma-Aldrich), or 50 U/ml catalase-polyethylene Downloaded from mature T cells isolated with DNeasy spin kits (Qiagen). Atg5 chimeras glycol (Sigma-Aldrich). were generated by transferring fetal liver cells into lethally irradiated con- genic recipient mice as described (35). All animal usage has been approved Quantitative PCR by the Duke University Institutional Animal Care and Use Committee. To determine mitochondrial DNA content in primary T cells, EasySep- ϩ Western blot enriched (StemCell Technologies) CD8 cells were sorted and DNA was extracted using a DNeasy kit (Qiagen). Quantitative PCR was performed

Total thymocytes, EasySep-enriched lymph node (LN)3 T cells (StemCell using a LightCycler FastStart DNA Master SYBR Green I kit on an iCycler http://www.jimmunol.org/ Technologies), and sorted TCR␤ϩCD4ϩ and TCR␤ϩCD8ϩ T cells from iQ real-time PCR detection system (Roche). Products from two sets of LNs were lysed in 50 mM Tris (pH 6.8), 10% glycerol, 2% SDS, and 100 mitochondrial DNA-specific primers (mtDNA F1, ACC ATT TGC AGA mM DTT. Membranes were blotted with Abs recognizing Atg7 (ProSci), CGC CAT AA; mtDNA R1, TGA AAT TGT TTG GGC TAC GG (40); Atg5 (Proteintech Group), cytochrome C (BD Pharmingen), Tom20 (Santa mtDNA F2, GCC CCA GAT ATA GCA TTC CC; mtDNA R2, GTT CAT Cruz Biotechnology), -inducing factor (AIF; Cell Signaling Tech- CCT GTT CCT GCT CC (41); actin DNA F, TGT TCC CTT CCA CAG nology), mtHsp70 (Affinity BioReagents), Bcl-2 (BD Pharmingen), Bcl-xL GGT GT; actin DNA R, TCC CAG TTG GTA ACA ATG CCA (41)) were (BD Pharmingen), Mcl-1 (Rockland), Bak (Upstate), Bax (eBioscience), normalized to a genomic actin cell loading control and analyzed using and actin (Santa Cruz Biotechnology). All blots except Bcl-2 were visu- relative expression software tool (REST) v2 software (42, 43). alized with anti-rabbit Alexa 680 (eBioscience), anti-mouse Alexa680 (eBioscience), and anti-goat Alexa 800 (Rockland) secondary Abs and read Results by guest on September 28, 2021 using an infrared imaging system (LI-COR Bioscience). Bcl-2 was visu- alized with HRP-conjugated anti-Armenian hamster (Jackson Immuno- Survival defect in Atg7-deficient mature T cells Research Laboratories) and Pico chemiluminescent substrate (Pierce). Previous work by our group (35) and others (34, 36, 37) has dem- Flow cytometry onstrated that autophagosomes form in primary T lymphocytes. Additionally, we have also demonstrated that the autophagy gene Single-cell suspensions of thymus, spleen, and LNs were lysed of RBC Atg5 contributes critically to the homeostatic survival of mouse T and incubated with FcR blocker (2.4G2; eBioscience). Cells were main- tained in PBS with 2% FBS on ice throughout staining before analysis. lymphocytes in vivo (35). As this autophagy protein also interacts Cells were stained with FITC, PE, PE-Cy5, allophycocyanin, and/or with FADD (Fas-associated death domain protein) and Bcl-xL (44, allophycocyanin-Cy7 anti-CD4, -CD8, -TCR␤, -CD44, -CD62L, 45), Atg5 may regulate T cell survival through its role in autopha- -CD25, -B220, and -CD45.2 (eBioscience, BioLegend, and BD Pharm- gosome formation or through interactions with extrinsic or intrin- ingen). Cell events were collected on a FACScan or FACStar, and data were analyzed using CellQuest (Becton Dickson) and FlowJo (Tree sic death pathways (46, 47). To determine whether the process of Star) software. was assayed using annexin V, 7-aminoacti- autophagy contributes to mature T cell survival, we examined the nomycin D, and propidium iodide staining (BD Pharmingen) or an ac- T cell compartment in a second autophagy-deficient genetic model tivated caspase 9 detection kit (Immunochemistry Technologies). To system. We crossed Atg7f/f mice (8) with mice expressing the T stain mitochondria, lymphocytes were incubated for 30 min at 37°C cell-specific Lck-Cre transgene (39). Consistent with the induction with 100 nM MitoTracker Green (Molecular Probes) in RPMI 1640 Ϫ Ϫ complete medium before surface Ab staining. For intracellular stains, of Lck-Cre expression at the CD4 CD8 double-negative (DN) cells were fixed in 2% PFA and permeabilized with 0.2% saponin stage of thymocyte development, we observed efficient genomic (Sigma-Aldrich) before staining with anti-Bcl-2 PE (BD Pharmingen) and protein deletion of Atg7 in double-positive (DP) and single- or anti-Mcl-1 (Rockland) followed by FITC anti-rabbit Abs (Jackson positive (SP) thymocytes as well as in mature CD4ϩ and CD8ϩ T ImmunoResearch Laboratories). lymphocytes in Atg7f/fLck-Cre mice (Fig. 1A). Additionally, we Transmission electron microscopy found impaired conjugation of Atg5 to Atg12 in both thymocytes Mature LN T cells after EasySep negative bead selection (StemCell Tech- and peripheral T cells lacking Atg7, an essential upstream enzyme nologies) were Ab stained and lightly fixed in 1% PFA. One to two million for this autophagic pathway conjugation process (Fig. 1B). These TCR␤ϩCD8ϩ T cells were sorted and fixed in a 4% glutaraldehyde 0.1 M data demonstrate that Atg7 is efficiently deleted, and they are con- sodium cacodylate buffer overnight. The samples were rinsed in 0.1 M sistent with a functional deficiency in autophagy in our model cacodylate buffer containing 7.5% sucrose three times for 15 min each and system. fixed in 1% osmium in cacodylate buffer for 1 h. After being washed three times in 0.11 M veronal acetate buffer for 15 min each, the samples were To determine the role of autophagy in T cell development and function, we examined thymocytes and mature T cells in Atg7f/f- Lck-Cre and Atg7f/f littermate controls. Although thymocyte de- 3 Abbreviations used in this paper: LN, lymph node; AIF, apoptosis-inducing factor; f/f DN, double negative; DP, double positive; MFI, mean fluorescence intensity; ROS, velopment appeared grossly normal in Atg7 Lck-Cre mice (Fig. reactive oxygen species; SP, single positive. 1C), pooled data from large numbers of mice showed a modest but 4048 AUTOPHAGY IN T CELL MITOCHONDRIA HOMEOSTASIS Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

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)by Western 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-Atg5 Western 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 CD8 Abs. 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 CD4ϩ or CD8ϩ T cells. E, Total cell numbers of thymocyte subsets, CD4ϩ T cells, CD8ϩ p Յ ,ء .(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 Ϯ SD .p Յ 0.0005 ,ءءء ;p Յ 0.005 ,ءء ;0.05 statistically significant decrease in CD4ϩ and CD8ϩ SP thymo- Atg7f/fLck-Cre mice (Fig. 1C). Atg7f/fLck-Cre mice also demon- cytes in these animals (Fig. 1E). In the spleen and lymph nodes, strated an increase in the relative percentage of CD44high there was a dramatic reduction in the percentage of T cells in CD62Llow peripheral T cells (Fig. 1D). This phenotype is most The Journal of Immunology 4049

FIGURE 2. Enhanced apoptotic death in mature Atg7f/fLck-Cre T cells. Flow cytometry analysis of freshly isolated Atg7f/f and Atg7f/fLck-Cre T cells for cell death. A, Percentage of dying (annexin Vϩ7- AADϪ) or dead (annexin Vϩ7-AADϩ) cells among gated CD4ϩ or CD8ϩ T cells. B, Percentage of CD4ϩ or CD8ϩ T cells with active intracellular caspase 9 by flow cytometry.

consistent with lymphopenia, as markers of cell activation includ- tem consistent with the selective elimination of Atg7 in T lin- ing CD69 and CD25 were not increased in these mice (Fig. 1D). eage cells (Fig. 1E). Ultimately, this resulted in a 75% decrease in total naive T cell Although numerous factors including modestly reduced thymo- numbers in peripheral Atg7-deficient T cells in secondary lym- cyte numbers (Fig. 1E) and alterations in the proliferative capacity phoid tissues (Fig. 1E). Finally, unlike Atg5Ϫ/Ϫ fetal liver chime- of autophagy-deficient T cells (38) may contribute to the defect in ras where total B lymphocyte numbers are decreased (35, 38), Atg7-deficient peripheral T cell homeostasis, mature T cells lack- we observed no reduction in B cell numbers in our model sys- ing this essential autophagy gene undergo enhanced apoptotic Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. Increased mitochondrial content in autophagy-deficient T cells. A, Flow cytometry analysis of MitoTracker Green mitochondrial staining in naive CD44lowAtg7f/f and Atg7f/fLck-Cre splenic T cells with MFI indicated. B, Mitochondrial content in Atg7-deficient peripheral lymphocytes as expressed by percentage of MitoTracker MFIs relative to control cells. Data were derived from four to six mice in three independent experiments (error bars, SD). C, MitoTracker and Tom20 colocalization in control and autophagy-deficient T cells. LN T cells were stained with MitoTracker Red and CD4-FITC, fixed, and permeabilized for intracellular staining with Tom20 and Alexa 350 anti-rabbit Ab. Confocal images were taken with the Zeiss ApoTome 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 ester perchlorate (TMRE) MFIs relative to control cells. Data were derived from three mice from two independent experiments (error bars, SE). E, Mitochondrial content in donor and host lymphocytes in Atg5-deficient and control fetal liver chimeras expressed as percentage of MitoTracker MFIs relative to control .p Յ 0.005 ,ءء p Յ 0.05 and ,ء .(cells. Data were derived in four to six mice from three independent experiments (error bars, SD 4050 AUTOPHAGY IN T CELL MITOCHONDRIA HOMEOSTASIS

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 MFI indicated. B and C, Mitochondrial content in Atg7 (B) or Atg5-deficient (C) thymocytes as expressed by per- centages of their MitoTracker MFIs relative to control cells. Data were derived from four to eight mice in three p Յ ,ءءء .(independent experiments (error bars, SD 0.0005. D, Comparison of MitoTracker Green staining between thymocytes and peripheral T cells by normal- f/f f/f izing Atg7 (filled symbols) and Atg7 Lck-Cre (open Downloaded from symbols) MFI to Atg7f/f DP fluorescence. Data from three to four mice from three independent experiments (error bars, SD). E, Mitochondrial content is unchanged during B lymphocyte development. Comparison of MitoTracker Green staining in developing bone marrow and peripheral wild-type B cells by normalizing MFI to pro-B cell fluorescence. Mitochondrial content was http://www.jimmunol.org/ measured by flow cytometry in CD43ϩB220ϩ pro-B cells, CD43ϪB220low pre-B cells, CD43ϪB220high im- mature B cells, and B220ϩ peripheral B cells in the spleen (Spl) and LN. Data are from three mice (error bars, SD). by guest on September 28, 2021

death. Freshly isolated mature Atg7f/fLck-Cre T cells displayed 3- deficient CD4ϩ and CD8ϩ T lymphocytes in the spleen and lymph to 4-fold enhanced rates of apoptosis ex vivo as measured by an- nodes (Fig. 3, A and B). As expected from this T cell-specific nexin V surface staining (Fig. 2A). Consistent with enhanced deletion model, we observed no difference in MitoTracker Green apoptotic death, freshly isolated Atg7-deficient T cells also staining of B220ϩ B lymphocytes (Fig. 3B). demonstrated enhanced caspase 9 activity by flow cytometry (Fig. Additionally, we confirmed this difference in the CD44low naive 2B). Taken together, these data demonstrate an important role for T cell subset since Atg7f/fLck-Cre mice have a relative increase in the essential autophagy gene Atg7 in maintaining peripheral T CD44highCD62Llow T cells consistent with a homeostatic response lymphocyte homeostasis. With previously published data from to lymphopenia (Fig. 3, A and B). We also examined whether Atg5-deficient chimeric mice, our results support a role for auto- MitoTracker Green staining measured by flow cytometry was spe- phagy in promoting mature T cell survival. cific for mitochondria by immunofluorescent microscopy. In both Atg7f/f and Atg7f/fLck-Cre T cells, MitoTracker stained a net- Enhanced mitochondrial content in Atg7-deficient mature T cells worked cytoplasmic structure that also stained positive for the The above results suggest that autophagy may have cytoprotective outer mitochondrial membrane marker Tom20 (Fig. 3C). Finally, functions in T lymphocytes. Given the central role of mitochondria we observed an increase in staining by the mitochondrial po- in lymphocyte apoptotic cell death (48) and the emerging role for tential- and volume-dependent dye TMRE in Atg7f/fLck-Cre T autophagy in maintaining proper homeostasis of this (6), cells when compared with Atg7f/f controls, consistent with an we investigated the mitochondrial compartment in autophagy-de- expansion of the functional mitochondrial content in these cells ficient T cells. We first examined the mitochondria in Atg7f/f and (Fig. 3D). Atg7f/fLck-Cre mature T cells using the relatively potential-inde- We next examined mitochondrial content in mature T cells lack- pendent mitochondria-specific vital dye MitoTracker Green (49). ing Atg5. In Atg5Ϫ/Ϫ fetal liver chimeric mice, CD45.2ϩ donor- MitoTracker Green is a lipophilic thiol-reactive dye that selec- derived CD4ϩ and CD8ϩ T cells from the spleen and lymph nodes tively labels mitochondrial inner membrane and matrix (50) and demonstrated dramatically enhanced MitoTracker Green staining has been used for measuring mitochondrial content in hematopoi- (Fig. 3E). No difference in MitoTracker Green staining in residual etic cells (51–53). Analysis by flow cytometry revealed a host T cells was observed in Atg5ϩ/ϩ and Atg5Ϫ/Ϫ chimeras (Fig. 50–150% increase in the MitoTracker Green staining of Atg7- 3E). Interestingly, Atg5Ϫ/Ϫ and control peripheral B lymphocytes The Journal of Immunology 4051 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. Mitochondrial morphology and DNA content in Atg7-deficient T cells. A, Representative transmission electron micrograph of Atg7f/f and Atg7f/fLck-Cre CD8ϩ T cell, with enlarged region showing mitochondrial morphology. B, Surface area of mitochondria in CD8ϩ T cell cross-sections. p Յ 0.05. C, Mitochondrial DNA ,ء .Values were calculated after manually outlining mitochondria and using the measure tool in AxioVision software content in Atg7-deficient CD8ϩ T cells. Quantitative PCR using two sets of primers specific for mitochondrial DNA was performed and normalized for input using a control genomic locus in sorted peripheral CD8ϩCD44low T cells from Atg7ϩ/ϩ and Atg7-deficient mice. Mitochondrial DNA in control T cells is 100%. Data are pooled from two independent experiments. D, Sample Western blot analysis of mitochondrial proteins in total thymocytes and sorted peripheral T cells from Atg7f/f and Atg7f/fLck-Cre T cells. Values listed below blot were normalized to actin, and wild-type CD4ϩ T cells were arbitrarily set to 100. E, Quantification of intensity of Western blots from multiple experiments. All values were normalized to actin, and CD4ϩ T cells were arbitrarily given the value of 100 in each experiment. Tom20 and CytC include three to six mice from three independent experiments; AIF and mtHsp70 include two to four mice from two independent experiments. had comparable MitoTracker Green staining, suggesting a T cell- mocytes. Although there was a significant increase in Mito- specific role for autophagy in mitochondrial homeostasis within Tracker Green mean fluorescent intensity (MFI) in Atg7- the lymphocyte compartment (Fig. 3E). deficient CD4ϩ and CD8ϩ SP thymocytes, the magnitude of this change (10–40%) was low when compared with mature Regulated mitochondrial content changes in developing T cells peripheral Atg7-deficient CD4ϩ and CD8ϩ T cells (Fig. 4, A To determine whether developing autophagy-deficient T cells and B). Additionally, we did not observe an increase in Mito- also contain enhanced mitochondrial content, we analyzed Mito- Tracker Green staining in Atg5Ϫ/Ϫ thymocytes when compared Tracker Green fluorescence in Atg7f/f and Atg7f/fLck-Cre thy- with Atg5ϩ/ϩ controls (Fig. 4C). The reason for this 4052 AUTOPHAGY IN T CELL MITOCHONDRIA HOMEOSTASIS Downloaded from

FIGURE 6. Increased ROS production and imbalanced expression of pro- and antiapoptotic proteins in Atg7-deficient T cells. A, ROS production in control and Atg7-deficient CD4ϩ 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 either dihydroethidium (DHE) or CM-H2DCFDA (DCF) and measuring mean cell fluorescence by flow cytometry. Data depicted are from two independent experiments for each detection reagent. B and D, Flow cytometry analysis of intracellular Bcl-2 and Mcl-1 expression in Atg7f/f (dark shaded histogram) http://www.jimmunol.org/ 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 line open 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 three independent experiments are pooled in graphical form as a percentage of Atg7-deficient over control indexed fluorescence. C and E, Western blot of sorted CD4ϩ and CD8ϩ T cells from Atg7f/f and Atg7f/fLck-Cre mice (Ͼ98% pure) for Bcl-2 and Mcl-1 expression. Data are representative of two independent experiments. F, Western blot of enriched Atg7-deficient and control spleen and lymph node T cells (Ն95% pure) for Bax and Bak. Numbers represent band intensity normalized to actin loading controls. Data are representative of two independent experiments.

discrepancy is unknown but may reflect subtle differences in the agy-deficient T cells exhibited clear outer membranes and cris- by guest on September 28, 2021 two model systems or the activity of Atg5 and Atg7 in T cell tae without specific evidence of dysfunction, including swelling autophagy. or condensation (Fig. 5A). When MitoTracker staining values from multiple mice were To determine whether differences observed in mitochondrial normalized to DP cell mean fluorescence, we observed a consistent content from MitoTracker Green staining might reflect differ- 50–75% reduction in the staining of wild-type T lymphocytes as ences in mitochondrial volume in these cells, we calculated they transitioned from the thymus to peripheral circulation (Fig. mitochondrial surface area in Ͼ40 cell cross-sections by man- ϩ ϩ 4D). This reduction was larger in CD8 than CD4 T cells, which ually outlining mitochondria using a quantification tool in Axio- may reflect the intermediate mitochondrial content phenotype in Vision (Zeiss). Large variations between individual cross-sec- CD8ϩ SP thymocytes (Fig. 4D). In contrast, mitochondrial content ϩ tions were found in all samples analyzed (Fig. 5B) and likely was not changed in developing B lymphocytes from CD43 reflect the uneven distribution of mitochondria in three-dimen- B220ϩ B cell precursors to peripheral mature B cell populations sional space within the cytoplasm of T cells observed by im- (Fig. 4E). When Atg7f/fLck-Cre MitoTracker Green values were munofluorescent microscopy. Nevertheless, a statistically sig- normalized to control DP fluorescence, it became clear that the nificant increase in mitochondrial surface in Atg7-deficient relative MFI increase in autophagy-deficient peripheral T lympho- CD8ϩ T cells was observed when compared with control T cells cytes largely reflects an impaired reduction in mitochondrial con- (Fig. 5B). To confirm this finding, we used a third measure of tent when compared with thymocytes (Fig. 4D). Thus, autophagy- dependent regulated changes in mitochondrial content mark the mitochondrial content in cells, mitochondrial DNA copy num- important developmental transition from thymocyte to circulating ber (40). By quantitative PCR analysis, we found that sorted f/f ϩ low ϳ peripheral T lymphocyte. Atg7 Lck-Cre CD8 CD44 T cells contained 2-fold more mitochondrial DNA than did control cells (Fig. 5C). Finally, we Mitochondrial morphology and DNA content in Atg7-deficient performed Western blots for a range of mitochondrial proteins T cells in Atg7f/f and Atg7f/fLck-Cre thymocytes and sorted T cells. Given the dramatic changes in mitochondrial content, particu- These results demonstrated a large increase in the inner-mem- larly in CD8ϩ Atg7f/fLck-Cre peripheral T cells, we sorted au- brane and intermembrane space proteins cytochrome C and tophagy-deficient and control CD8ϩ T cells to visually exam- AIF, with less dramatic changes in the outer mitochondrial ined mitochondria by transmission electron microscopy. transport protein Tom20 and inner matrix chaperone mtHsp70 Although there was notable variation in mitochondrial morphol- (Fig. 5, D and E). Taken together, these results suggest that in ogy between individual cell cross-sections, the mitochondrial the absence of autophagy increased numbers of grossly normal shape and appearance in Atg7f/fLck-Cre T cells were largely mitochondria are present in mature T cells and that autophagy comparable to controls (Fig. 5A). The mitochondria in autoph- is critical to clear mitochondria in these cells. The Journal of Immunology 4053

Increased ROS production and imbalanced expression of regulated. A reduction of mitochondrial content marks the transi- pro- and antiapoptotic proteins in Atg7-deficient T lymphocytes tion from thymocytes to peripheral mature T cells. Third, the clear- Mitochondria are responsible for numerous important metabolic ance of superfluous mitochondria in mature T cells critically de- pathways within eukaryotic cells. To determine whether increased pends on autophagy. This is supported by our measurement of mitochondrial content in autophagy-deficient T cells was associ- mitochondrial content through FACS analysis of MitoTracker ated with functional changes in these cells, we examined ROS Green-stained mitochondria, direct survey of mitochondrial sur- production in cultured T lymphocytes. We observed a 2-fold in- face area under transmission electron microscopy, quantitative crease in ROS production by Atg7-deficient T cells as measured by determination of mitochondrial DNA, as well as the expression dihydroethidium and CM-H2DCFDA staining (Fig. 6A). ROS pro- levels of various proteins associated with mitochondria. Interest- duction has been shown to play a proapoptotic role in T cell hy- ingly, this phenomenon appears to be limited to T lymphocytes, as bridomas as well as in activated primary T cells (54, 55). mitochondrial content is not developmentally regulated in B lym- In addition to producing ROS, mitochondria can become toxic phocytes. Fourth, in the absence of autophagy, we have observed to cells through their role in the intrinsic death pathway. Death not only enhanced mitochondrial content but also abnormality in initiated through the mitochondria is largely regulated by Bcl-2 mitochondria-associated functions, including ROS production and family proteins and classically results in the release of proapop- apoptosis. totic factors normally sequestered away from downstream effectors The precise role of autophagy in cell survival and cell death in the mitochondrial intermembrane space (48). To determine remains controversial, and this controversy likely reflects the com- whether abnormalities in Bcl-2 family protein expression are as- plex functional and molecular intersection of autophagy with pro- Downloaded from sociated with enhanced apoptosis in autophagy-deficient mature T survival and pro-death pathways (46, 59, 60). In cells where apo- lymphocytes, we first examined the expression of the two major ptosis is inhibited, the degradation of cytoplasmic material in antiapoptotic Bcl-2 family members expressed in naive T cells, autophagosomes protects cells from death by providing essential Bcl-2 and Mcl-1 (56, 57). The expression of Bcl-2 as assessed by metabolic support during periods of nutrient deprivation (9); how- f/f both flow cytometry and Western blot was increased in Atg7 Lck- ever, the autophagic degradation of essential proteins, including Cre T cells (Fig. 6, B and C). Conversely, we found no significant

catalase (61, 62), and organelles (20) can also commit theses cell http://www.jimmunol.org/ f/f increase in Mcl-1 expression in Atg7 Lck-Cre cells as measured to death. Although the outcome of autophagosome formation in by both flow cytometry and Western blot (Fig. 6, D and E). We cell survival may depend on cell type and context, autophagy observed low levels of Bcl-xL expression as assessed by Western clearly serves a critical role in the homeostatic survival of cells in blot in both Atg7-deficient and control T cells, consistent with the vivo. This has been most clearly demonstrated in the CNS, where low expression of this antiapoptotic protein in naive T lympho- the conditional deletion of the essential autophagy genes Atg5 or cytes (unpublished data). Thus, T cells lacking the essential auto- Atg7 leads to associated with an increase in phagy gene Atg7 demonstrate a selective up-regulation of Bcl-2, TUNEL-positive apoptotic cells (10, 11). Our results here, together but not other antiapoptotic family members. with our previous data (35), demonstrate that deletion of Atg5 or

We next examined the proapoptotic members of the Bcl-2 family, by guest on September 28, 2021 Atg7 in primary mouse T lymphocytes leads to a dramatic impair- Bax and Bak. While Bax protein levels remained unchanged in au- ment of naive T cell peripheral survival with up to a 75% reduction tophagy-deficient T lymphocytes, Bak protein was increased ϳ2-fold by Western blot analysis (Fig. 6F). Additionally, consistent with the in the number of circulating T cells. Thus autophagy constitutes a increase in mitochondrial content in our autophagy-deficient T cells, novel pro-survival pathway in mature T lymphocytes. we found a significant increase in death-inducing mitochondrial pro- Our finding that mitochondrial content is developmentally teins cytochrome c and AIF expression in Atg7f/fLck-Cre T cells regulated in T lymphocytes and the reduction of mitochondrial when compared with Atg7f/f controls (Fig. 5, D and E). Given the content in peripheral T cells critically depends on autophagy central role of the BH3 only protein Bim in the induction of naive and has identified a novel mechanism by which mature T lympho- activated T cell death (58), we also examined Bim expression but cyte homeostasis is maintained. Thymocytes from DN, DP, and ϩ observed no difference between autophagy-deficient and control T CD4 SP compartments contain high content of mitochondria. ϩ cells (unpublished data). Taken together, these data suggest that In contrast, mitochondrial content in CD8 SP thymocytes is perturbations in the balance of pro- and antiapoptotic proteins may reduced to an intermediate level. Furthermore, although mito- contribute to cell death in primary T cells in the absence of autophagy. chondrial content in CD4ϩ mature T cells is reduced by ϳ50% when compared with DP thymocytes, this reduction is ϳ80% ϩ Discussion for CD8 mature T cells. This more dramatic reduction of mi- tochondrial content in developing CD8ϩ T cells may reflect Although autophagy has long been recognized, its functions in their lower tolerance for superfluous mitochondria than in various physiological and pathological processes have only begun CD4ϩ mature T lymphocytes. Consistent with this notion, to be elucidated. Our studies have revealed several key findings ϩ regarding the role of autophagy in regulating T lymphocyte sur- CD8 T cells lacking Atg5 (35) or Atg7 exhibit higher apo- ptosis rates and more dramatic reductions in cell numbers than vival. First, our data strongly suggest that autophagy itself is es- ϩ sential for mature T cell survival. Although our previous results do autophagy-deficient CD4 T cells. Interestingly, although show that Atg5-deficient mature T cells undergo massive apoptosis mitochondrial content is increased, morphology appears normal (35), it was not clear whether the defective survival of Atg5-defi- under transmission electron microscopy. This is in contrast to cient T cells was caused by the role of Atg5 in the induction of the deformed mitochondria in Atg7-deficient hepatocytes (8). autophagy or other survival pathways, as Atg5 interacts with This difference may suggest a role of autophagy in removing

FADD and Bcl-xL (44, 45). The fact that T lymphocytes lacking a superfluous mitochondria in T cells and damaged mitochondria second essential autophagy gene, Atg7, exhibit a similar survival in liver cells. Alternatively, low levels of mitochondrial damage defect to that of Atg5-deficient T cells strongly argues for a role for may result in the rapid apoptosis of T lymphocytes, making autophagy in maintaining mature T cell survival. Second, we show abnormal mitochondria more difficult to detect among live au- that mitochondrial content in T lymphocytes is developmentally tophagy-deficient T cells. 4054 AUTOPHAGY IN T CELL MITOCHONDRIA HOMEOSTASIS

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