Macrophages Rapidly Transfer Pathogens from Lipid Raft Vacuoles to Autophagosomes

Macrophages activate autophagy as an immediate response to Legionella pneumophila infection, but what marks the pathogen phagosome as a target for the autophagy machinery is not known. Because a variety of bacteria, parasites, viruses, and toxins that associate with the endoplasmic reticulum enter host cells by a cholesterol-dependent route, we tested the hypothesis that autophagy is triggered when microbes engage components of lipid raft domains. As the intracellular respiratory pathogen L. pneumophila or the extracellular uropathogen FimH⁺ Escherichia coli entered macrophages by a cholesterol-sensitive mechanism, they immediately resided in vacuoles rich in glycosylphosphatidylinositol moieties and the autophagy enzyme Atg7. As expected for autophagosomes, the vacuoles sequentially acquired the endoplasmic reticulum protein BiP, the autophagy markers Atg8 and monodansyl-cadaverine, and the lysosomal protein LAMP-1. A robust macrophage response to the pathogens was cholesterol-dependent, since fewer Atg7-rich vacuoles were observed when macrophages were pre-treated with methyl-beta-cyclodextrin or filipin. A model in which macrophages exploit autophagy to capture pathogens within the lipid raft pathway for antigen presentation prior to disposal in lysosomes is discussed.     

Autophagy is an immediate macrophage response to Legionella pneumophila

After ingestion by macrophages, Legionella pneumophila enter spacious vacuoles that are quickly enveloped by endoplasmic reticulum (ER), then slowly transferred to lysosomes. Here we demonstrate that the macrophage autophagy machinery recognizes the pathogen phagosome as cargo for lysosome delivery. The autophagy conjugation enzyme Atg7 immediately translocated to phagosomes harbouring virulent Legionella. Subsequently, Atg8, a second autophagy enzyme, and monodansyl-cadaverine (MDC), a dye that accumulates in acidic autophagosomes, decorated the pathogen vacuoles. The autophagy machinery responded to 10-30 kDa species released into culture supernatants by Type IV secretion-competent Legionella, as judged by the macrophages' processing of Atg8 and formation of vacuoles that sequentially acquired Atg7, Atg8 and MDC. When compared with autophagosomes stimulated by rapamycin, Legionella vacuoles acquired Atg7, Atg8 and MDC more slowly, and Atg8 processing was also delayed. Moreover, compared with autophagosomes of Legionella-permissive naip5 mutant A/J macrophages, those of resistant C57BL/6 J macrophages matured quickly, preventing efficient Legionella replication. Accordingly, we discuss a model in which macrophages elevate autophagy as a barrier to infection, a decision influenced by regulatory interactions between Naip proteins and caspases.     

Lipid droplet autophagy in the yeast Saccharomyces cerevisiae

Cytosolic lipid droplets (LDs) are ubiquitous organelles in prokaryotes and eukaryotes that play a key role in cellular and organismal lipid homeostasis. Triacylglycerols (TAGs) and steryl esters, which are stored in LDs, are typically mobilized in growing cells or upon hormonal stimulation by LD-associated lipases and steryl ester hydrolases. Here we show that in the yeast Saccharomyces cerevisiae, LDs can also be turned over in vacuoles/lysosomes by a process that morphologically resembles microautophagy. A distinct set of proteins involved in LD autophagy is identified, which includes the core autophagic machinery but not Atg11 or Atg20. Thus LD autophagy is distinct from endoplasmic reticulum–autophagy, pexophagy, or mitophagy, despite the close association between these organelles. Atg15 is responsible for TAG breakdown in vacuoles and is required to support growth when de novo fatty acid synthesis is compromised. Furthermore, none of the core autophagy proteins, including Atg1 and Atg8, is required for LD formation in yeast.     

Immunity to vacuolar pathogens: What can we learn from Legionella?

Intracellular pathogens can manipulate host cellular pathways to create specialized organelles. These pathogen-modified vacuoles permit the survival and replication of bacterial and protozoan microorganisms inside of the host cell. By establishing an atypical organelle, intracellular pathogens present unique challenges to the host immune system. To understand pathogenesis, it is important to not only investigate how these organisms create unique subcellular compartments, but to also determine how mammalian immune systems have evolved to detect and respond to pathogens sequestered in specialized vacuoles. Recent studies have identified genes in the respiratory pathogen Legionella pneumophila that are essential for establishing a unique endoplasmic reticulum-derived organelle inside of mammalian macrophages, making this pathogen an attractive model system for investigations on host immune responses that are specific for bacteria that establish vacuoles disconnected from the endocytic pathway. This review will focus on the host immune response to Legionella and highlight areas of Legionella research that should help elucidate host strategies to combat infections by intracellular pathogens.     

Autophagy deficiency in myeloid cells increases susceptibility to obesity-induced diabetes and experimental colitis

Autophagy, which is critical for the proper turnover of organelles such as endoplasmic reticulum and mitochondria, affects diverse aspects of metabolism, and its dysregulation has been incriminated in various metabolic disorders. However, the role of autophagy of myeloid cells in adipose tissue inflammation and type 2 diabetes has not been addressed. We produced mice with myeloid cell-specific deletion of Atg7 (autophagy-related 7), an essential autophagy gene (Atg7 conditional knockout [cKO] mice). While Atg7 cKO mice were metabolically indistinguishable from control mice, they developed diabetes when bred to ob/w mice (Atg7 cKO-ob/ob mice), accompanied by increases in the crown-like structure, inflammatory cytokine expression and inflammasome activation in adipose tissue. Mφs (macrophages) from Atg7 cKO mice showed significantly higher interleukin 1 β release and inflammasome activation in response to a palmitic acid plus lipopolysaccharide combination. Moreover, a decrease in the NAD⁺:NADH ratio and increase in intracellular ROS content after treatment with palmitic acid in combination with lipopolysaccharide were more pronounced in Mφs from Atg7 cKO mice, suggesting that mitochondrial dysfunction in autophagy-deficient Mφs leads to an increase in lipid-induced inflammasome and metabolic deterioration in Atg7 cKO-ob/ob mice. Atg7 cKO mice were more susceptible to experimental colitis, accompanied by increased colonic cytokine expression, T helper 1 skewing and systemic bacterial invasion. These results suggest that autophagy of Mφs is important for the control of inflammasome activation in response to metabolic or extrinsic stress, and autophagy deficiency in Mφs may contribute to the progression of metabolic syndrome associated with lipid injury and colitis.     

Autophagy and inflammatory cell death, partners of innate immunity

By law in the evolutionary jungle, any host defense mechanism that efficiently kills microbes also exerts a strong selective pressure for tolerant variants to emerge. As a consequence, pathogens can be exploited as powerful tools to examine host defense mechanisms. Recent studies of the confrontation between macrophages and the opportunistic pathogen Legionella pneumophila have revealed a regulatory mechanism that may link autophagy to pyroptosis, a type of programmed cell death. Building from the extensive literature on autophagy, cell death, and innate immunity, we propose here a testable model in which the NOD-LRR protein Naip5 dictates whether murine macrophages elevate autophagy or pyroptosis as a barrier to infection.     

Macroautophagy is dispensable for intracellular replication of Legionella pneumophila in Dictyostelium discoideum

The Gram-negative bacterium Legionella pneumophila is a facultative intracellular pathogen of free-living amoebae and mammalian phagocytes. L. pneumophila is engulfed in phagosomes that initially avoid fusion with lysosomes. The phagosome associates with endoplasmic reticulum (ER) and mitochondria and eventually resembles ER. The morphological similarity of the replication vacuole to autophagosomes, and enhanced bacterial replication in response to macroautophagy-inducing starvation, led to the hypothesis that L. pneumophila infection requires macroautophagy. As L. pneumophila replicates in Dictyostelium discoideum, and macroautophagy genes have been identified and mutated in D. discoideum, we have taken a genetic and cell biological approach to evaluate the relationship between host macroautophagy and intracellular replication of L. pneumophila. Mutation of the apg1, apg5, apg6, apg7 and apg8 genes produced typical macroautophagy defects, including reduced bulk protein degradation and cell viability during starvation. We show that L. pneumophila replicates normally in D. discoideum macroautophagy mutants and produces replication vacuoles that are morphologically indistinguishable from those in wild-type D. discoideum. Furthermore, a green fluorescent protein (GFP)-tagged marker of autophagosomes, Apg8, does not systematically co-localize with DsRed-labelled L. pneumophila. We conclude that macroautophagy is dispensable for L. pneumophila intracellular replication in D. discoideum.     

Lipid raft-dependent uptake, signalling and intracellular fate of Porphyromonas gingivalis in mouse macrophages

Lipid rafts are cholesterol-enriched microdomains involved in cellular trafficking and implicated as portals for certain pathogens. We sought to determine whether the oral pathogen Porphyromonas gingivalis enters macrophages via lipid rafts, and if so, to examine the impact of raft entry on its intracellular fate. Using J774A.1 mouse macrophages, we found that P. gingivalis colocalizes with lipid rafts in a cholesterol-dependent way. Depletion of cellular cholesterol using methyl-beta-cyclodextrin resulted in about 50% inhibition of P. gingivalis uptake, although this effect was reversed by cholesterol reconstitution. The intracellular survival of P. gingivalis was dramatically inhibited in cholesterol-depleted cells relative to untreated or cholesterol-reconstituted cells, even when infections were adjusted to allow equilibration of the initial intracellular bacterial load. P. gingivalis thus appeared to exploit raft-mediated uptake for promoting its survival. Consistent with this, lipid raft disruption enhanced the colocalization of internalized P. gingivalis with lysosomes. In contrast, raft disruption did not affect the expression of host receptors interacting with P. gingivalis, although it significantly inhibited signal transduction. In summary, P. gingivalis uses macrophage lipid rafts as signalling and entry platforms, which determine its intracellular fate to the pathogen's own advantage.     

A life-span extending form of autophagy employs the vacuole-vacuole fusion machinery

While autophagy is believed to be beneficial for life-span extension, it is controversial which forms or aspects of autophagy are responsible for this effect. We addressed this topic by analyzing the life span of yeast autophagy mutants under caloric restriction, a longevity manipulation. Surprisingly, we discovered that the majority of proteins involved in macroautophagy and several forms of microautophagy were dispensable for life-span extension. The only autophagy protein that is critical for life-span extension was Atg15, a lipase that is located in the endoplasmic reticulum (ER) and transported to vacuoles for disintegrating membranes of autophagic bodies. We further found that vacuole-vacuole fusion was required for life-span extension, which was indicated by the shortened life span of mutants missing proteins (ypt7Delta, nyv1Delta, vac8Delta) or lipids (erg6Delta) involved in fusion. Since a known function of vacuole-vacuole fusion is the maintenance of the vacuole membrane integrity, we analyzed aged vacuoles and discovered that aged cells had altered vacuolar morphology and accumulated autophagic bodies, suggesting that certain forms of autophagy do contribute to longevity. Like aged cells, erg6Delta accumulated autophagic bodies, which is likely caused by a defect in lipase instead of proteases due to the existence of multiple vacuolar proteases. Since macroautophagy is not blocked by erg6Delta, we propose that a new form of autophagy transports Atg15 via the fusion of vacuoles with vesicles derived from ER, and we designate this putative form of autophagy as secretophagy. Pending future biochemical studies, the concept of secretophagy may provide a mechanism for autophagy in life-span extension.     

Autophagy facilitates secretion and protects against degeneration of the Harderian gland

The epithelial derived Harderian gland consists of 2 types of secretory cells. The more numerous type A cells are responsible for the secretion of lipid droplets, while type B cells produce dark granules of multilamellar bodies. The process of autophagy is constitutively active in the Harderian gland, as confirmed by our analysis of LC3 processing in GFP-LC3 transgenic mice. This process is compromised by epithelial deletion of Atg7. Morphologically, the Atg7 mutant glands are hypotrophic and degenerated, with highly vacuolated cells and pyknotic nuclei. The mutant glands accumulate lipid droplets coated with PLIN2 (perilipin 2) and contain deposits of cholesterol, ubiquitinated proteins, SQSTM1/p62 (sequestosome 1) positive aggregates and other metabolic products such as porphyrin. Immunofluorescence stainings show that distinct cells strongly aggregate both proteins and lipids. Electron microscopy of the Harderian glands reveals that its organized structure is compromised, and the presence of large intracellular lipid droplets and heterologous aggregates. We attribute the occurrence of large vacuoles to a malfunction in the formation of multilamellar bodies found in the less abundant type B Harderian gland cells. This defect causes the formation of large tertiary lysosomes of heterologous content and is accompanied by the generation of tight lamellar stacks of endoplasmic reticulum in a pseudo-crystalline form. To test the hypothesis that lipid and protein accumulation is the cause for the degeneration in autophagy-deficient Harderian glands, epithelial cells were treated with a combination of the proteasome inhibitor and free fatty acids, to induce aggregation of misfolded proteins and lipid accumulation, respectively. The results show that lipid accumulation indeed enhanced the toxicity of misfolded proteins and that this was even more pronounced in autophagy-deficient cells. Thus, we conclude autophagy controls protein and lipid catabolism and anabolism to facilitate bulk production of secretory vesicles of the Harderian gland.     

Autophagy facilitates secretion and protects against degeneration of the Harderian gland

The epithelial derived Harderian gland consists of 2 types of secretory cells. The more numerous type A cells are responsible for the secretion of lipid droplets, while type B cells produce dark granules of multilamellar bodies. The process of autophagy is constitutively active in the Harderian gland, as confirmed by our analysis of LC3 processing in GFP-LC3 transgenic mice. This process is compromised by epithelial deletion of Atg7. Morphologically, the Atg7 mutant glands are hypotrophic and degenerated, with highly vacuolated cells and pyknotic nuclei. The mutant glands accumulate lipid droplets coated with PLIN2 (perilipin 2) and contain deposits of cholesterol, ubiquitinated proteins, SQSTM1/p62 (sequestosome 1) positive aggregates and other metabolic products such as porphyrin. Immunofluorescence stainings show that distinct cells strongly aggregate both proteins and lipids. Electron microscopy of the Harderian glands reveals that its organized structure is compromised, and the presence of large intracellular lipid droplets and heterologous aggregates. We attribute the occurrence of large vacuoles to a malfunction in the formation of multilamellar bodies found in the less abundant type B Harderian gland cells. This defect causes the formation of large tertiary lysosomes of heterologous content and is accompanied by the generation of tight lamellar stacks of endoplasmic reticulum in a pseudo-crystalline form. To test the hypothesis that lipid and protein accumulation is the cause for the degeneration in autophagy-deficient Harderian glands, epithelial cells were treated with a combination of the proteasome inhibitor and free fatty acids, to induce aggregation of misfolded proteins and lipid accumulation, respectively. The results show that lipid accumulation indeed enhanced the toxicity of misfolded proteins and that this was even more pronounced in autophagy-deficient cells. Thus, we conclude autophagy controls protein and lipid catabolism and anabolism to facilitate bulk production of secretory vesicles of the Harderian gland.     

Autophagy regulates endoplasmic reticulum homeostasis and calcium mobilization in T lymphocytes

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved intracellular bulk degradation pathway that plays critical roles in eliminating intracellular pathogens, presenting endogenous Ags, and regulating T lymphocyte survival and proliferation. In this study, we have investigated the role of autophagy in regulating the endoplasmic reticulum (ER) compartment in T lymphocytes. We found that ER content is expanded in mature autophagy-related protein (Atg) 7-deficient T lymphocytes. Atg7-deficient T cells stimulated through the TCR display impaired influx, but not efflux, of calcium, and ER calcium stores are increased in Atg7-deficient T cells. Treatment with the ER sarco/ER Ca(2+)-ATPase pump inhibitor thapsigargin rescues the calcium influx defect in Atg7-deficient T lymphocytes, suggesting that this impairment is caused by an intrinsic defect in ER. Furthermore, we found that the stimulation-induced redistribution of stromal interaction molecule-1, a critical event for the store-operated Ca(2+) release-activated Ca(2+) channel opening, is impaired in Atg7-deficient T cells. Together, these findings indicate that the expanded ER compartment in Atg7-deficient T cells contains increased calcium stores, and the inability of these stores to be depleted causes defective calcium influx in these cells. Our results demonstrate that autophagy plays an important role in maintaining ER and calcium homeostasis in T lymphocytes.     

Endoplasmic reticulum stress: a new pathway to induce autophagy

Autophagy is a response to the stress of nutrient limitation in yeast, whereby cytosolic long-lived proteins and organelles are nonselectively degraded, and the resulting macromolecules are recycled to allow new protein synthesis that is essential for survival. We recently revealed that endoplasmic reticulum (ER) stress induces autophagy. When misfolded proteins accumulate in the ER the resulting stress activates the unfolded protein response (UPR) to induce the expression of chaperones and proteins involved in the recovery process. Under conditions of ER stress, the preautophagosomal structure is assembled, and transport of autophagosomes to the vacuole is stimulated in an Atg protein-dependent manner. Interestingly, Atg1 has high kinase activity during ER stress-induced autophagy similar to the situation in starvation-induced autophagy.     

Atg2: A novel phospholipid transfer protein that mediates de novo autophagosome biogenesis

The degradation of cytoplasmic components via autophagy is crucial for intracellular homeostasis. In the process of autophagy, a newly synthesized isolation membrane (IM) is developed to sequester degradation targets and eventually the IM seals, forming an autophagosome. One of the most poorly understood autophagy‐related proteins is Atg2, which is known to localize to a contact site between the edge of the expanding IM and the exit site of the endoplasmic reticulum (ERES). Recent advances in structural and biochemical analyses have been applied to Atg2 and have revealed it to be a novel multifunctional protein that tethers membranes and transfers phospholipids between them. Considering that Atg2 is essential for the expansion of the IM that requires phospholipids as building blocks, it is suggested that Atg2 transfers phospholipids from the ERES to the IM during the process of autophagosome formation, suggesting that lipid transfer proteins can mediate de novo organelle biogenesis.     

Toll-like receptors control autophagy

Autophagy is a newly recognized innate defense mechanism, acting as a cell-autonomous system for elimination of intracellular pathogens. The signals and signalling pathways inducing autophagy in response to pathogen invasion are presently not known. Here we show that autophagy is controlled by recognizing conserved pathogen-associated molecular patterns (PAMPs). We screened a PAMP library for effects on autophagy in RAW 264.7 macrophages and found that several prototype Toll-like receptor (TLR) ligands induced autophagy. Single-stranded RNA and TLR7 generated the most potent effects. Induction of autophagy via TLR7 depended on MyD88 expression. Stimulation of autophagy with TLR7 ligands was functional in eliminating intracellular microbes, even when the target pathogen was normally not associated with TLR7 signalling. These findings link two innate immunity defense systems, TLR signalling and autophagy, provide a potential molecular mechanism for induction of autophagy in response to pathogen invasion, and show that the newly recognized ability of TLR ligands to stimulate autophagy can be used to treat intracellular pathogens.     

Proteome analysis of Legionella vacuoles purified by magnetic immunoseparation reveals secretory and endosomal GTPases

Legionella pneumophila , the causative agent of Legionnaires' disease, replicates in macrophages and amoebae within ' Legionella -containing vacuoles' (LCVs), which communicate with the early secretory pathway and the endoplasmic reticulum. Formation of LCVs requires the bacterial Icm/Dot type IV secretion system. The Icm/Dot-translocated effector protein SidC selectively anchors to LCVs by binding the host lipid phosphatidylinositol-4-phosphate (PtdIns(4) P ). Here, we describe a novel and simple approach to purify intact vacuoles formed by L. pneumophila within Dictyostelium discoideum by using magnetic immunoseparation with an antibody against SidC, followed by density gradient centrifugation. To monitor LCV purification by fluorescence microscopy, we used Dictyostelium producing the LCV marker calnexin-GFP and L. pneumophila labeled with the red fluorescent protein DsRed. A proteome analysis of purified LCVs by liquid chromatography coupled to tandem mass spectrometry revealed 566 host proteins, including known LCV components, such as the small GTPases Arf1, Rab1 and Rab7. Rab8, an endosomal regulator of the late secretory pathway originating from the trans Golgi network, and the endosomal GTPase Rab14 were identified as novel LCV components, which were found to be present on vacuoles harboring wild-type but not Icm/Dot-deficient L. pneumophila . Thus, LCVs also communicate with the late secretory and endosomal pathways. Depletion of Rab8 or Arf1 by RNA interference reduced the amount of SidC on LCVs, indicating that the GTPases promote the recruitment of Legionella effectors by regulating the level of PtdIns(4) P .     

Autophagy and Inflammatory Cell Death,Partners of Innate Immunity

Alas, it is the law of the evolutionary jungle that any host defen! se mechanism that efficiently kills microbes also exerts a strong selective pressure for tolerant variants to emerge. A silver lining in this otherwise dark cloud is that pathogens can be exploited as powerful tools to examine host defense mechanisms. Recent studies of the confrontation between macrophages and the opportunistic pathogen Legionella pneumophila have revealed a regulatory mechanism that may link autophagy to pyroptosis. Building from the extensive literature on autophagy, cell death, and innate immunity, we propose here a testable model in which the NOD-LRR protein Naip5 dictates whether macrophages elevate autophagy or pyroptosis as a barrier to infection.     

Shiga toxins induce autophagy leading to differential signalling pathways in toxin-sensitive and toxin-resistant human cells

The bacterial virulence factors Shiga toxins (Stxs) are expressed by Shigella dysenteriae serotype 1 and certain Escherichia coli strains. Stxs are protein synthesis inhibitors and induce apoptosis in many cell types. Stxs induce apoptosis via prolonged endoplasmic reticulum stress signalling to activate both extrinsic and intrinsic pathways in human myeloid cells. Studies have shown that autophagy, a lysosome-dependent catabolic process, may be associated with activation of pro-survival or death processes. It is currently unknown if autophagy contributes to apoptosis or protects cells from Stxs. To study cellular responses to Stxs, we intoxicated toxin-sensitive cells (THP-1 and HK-2 cells), and toxin-resistant cells (primary human monocyte-derived macrophages) and examined toxin intracellular trafficking and autophagosome formation. Stxs translocated to different cell compartments in toxin-resistant versus toxin-sensitive cells. Confocal microscopy revealed autophagosome formation in both toxin-resistant and toxin-sensitive cells. Proteolytic cleavage of Atg5 and Beclin-1 plays pivotal roles in switching non-cytotoxic autophagy to cell death signalling. We detected cleaved forms of Atg5 and Beclin-1 in Stx-treated toxin-sensitive cells, while cleaved caspases, calpains, Atg5 and Beclin-1 were not detected in toxin-resistant primary human monocytes and macrophages. These findings suggest that toxin sensitivity correlates with caspase and calpain activation, leading to Atg5 and Beclin-1 cleavage.     

Lipid droplet–mediated ER homeostasis regulates autophagy and cell survival during starvation

Lipid droplets (LDs) are conserved organelles for intracellular neutral lipid storage. Recent studies suggest that LDs function as direct lipid sources for autophagy, a central catabolic process in homeostasis and stress response. Here, we demonstrate that LDs are dispensable as a membrane source for autophagy, but fulfill critical functions for endoplasmic reticulum (ER) homeostasis linked to autophagy regulation. In the absence of LDs, yeast cells display alterations in their phospholipid composition and fail to buffer de novo fatty acid (FA) synthesis causing chronic stress and morphologic changes in the ER. These defects compromise regulation of autophagy, including formation of multiple aberrant Atg8 puncta and drastically impaired autophagosome biogenesis, leading to severe defects in nutrient stress survival. Importantly, metabolically corrected phospholipid composition and improved FA resistance of LD-deficient cells cure autophagy and cell survival. Together, our findings provide novel insight into the complex interrelation between LD-mediated lipid homeostasis and the regulation of autophagy potentially relevant for neurodegenerative and metabolic diseases.     

Temporal regulation of intracellular organelle homeostasis in T lymphocytes by autophagy

The highly conserved self-degradation pathway known as autophagy plays important roles in regulating T lymphocyte homeostasis. Recently, we found that T lymphocytes lacking the autophagy-related gene Atg5 or Atg7 have defective survival and contain expanded mitochondria and endoplasmic reticulum (ER); however, whether these defects are caused by impaired autophagy or by defects in their autophagy-independent signaling capacity of Atg5 or Atg7 in T lymphocytes remains unknown. Furthermore, the function of the microtubule-associated protein L chain 3 (LC3) conjugation system in T lymphocytes remains unclear. To address these questions, we generated conditional knockout mice with specific deletion of Atg3, a ubiquitin enzyme E2-like molecule involved in the LC3 conjugation system, in T lymphocytes. Atg3-deficient T lymphocytes displayed a phenotype similar to those of Atg7- and Atg5-deficient T cells. The survival of Atg3-deficient naive CD4(+) and CD8(+) T cells was defective. Furthermore, the mitochondria and ER were expanded in Atg3-deficient T cells. Interestingly, mitochondrial and ER content did not change instantly upon inducible deletion of Atg3 in mature T lymphocytes in vitro. Instead, it began to expand 10 d after inducible deletion of Atg3 in mature T lymphocytes, and mitochondrial content continued to increase on day 18. Cell death began to increase 24 d after inducible deletion of Atg3. These data show that the LC3 conjugation system is essential for autophagy in T lymphocytes. Our data suggest that autophagy promotes T lymphocyte survival by regulating organelle homeostasis and that the decreased survival of autophagy-deficient T cells is due to the temporal accumulation of these autophagy-related defects.