REVIEW

Autophagy and autophagy-related proteins

in the immune system
Shusaku T Shibutani1,2,5, Tatsuya Saitoh3, Heike Nowag4, Christian Mnz4 & Tamotsu Yoshimori1,2

Autophagy is an intracellular bulk degradation system that is highly conserved in eukaryotes. The discovery of autophagy-related
('ATG') proteins in the 1990s greatly advanced the mechanistic understanding of autophagy and clarified the fact that autophagy
serves important roles in various biological processes. In addition, studies have revealed other roles for the autophagic machinery
2015 Nature America, Inc. All rights reserved.

beyond autophagy. In this Review, we introduce advances in the knowledge of the roles of autophagy and its components in
immunity, including innate immunity, inflammatory responses and adaptive immunity.

In macroautophagy (called simply autophagy here), a double-mem-
brane organelle called the autophagosome has a central role. The
formation of autophagosomes is maintained at relatively low levels
under non-stress conditions but is induced substantially by various
cellular stresses (for example, amino acid starvation). The forma-
tion of autophagosomes starts with the emergence of a membrane
cup called the isolation membrane (or phagophore) (Fig. 1). The
isolation membrane elongates and closes, which results in the forma-
tion of a double-membranous autophagosome that engulfs a portion
of the cytoplasm. Then, subsequent autophagosome-lysosome fusion
leads to the digestion of its contents and inner membrane by lysosomal
hydrolases (Fig. 1).
A growing body of evidence suggests that autophagy has important
roles in various biological processes at cellular and physiological levels
and is generally beneficial to cells and whole organisms1. Furthermore,
npg
related genes in the 1990s (refs. 24). 40 genes encoding autophagy-
related (ATG) proteins have been identified so far, and the 15 core
genes encoding ATG proteins in yeast (ATG1ATG10, ATG12ATG14,
ATG16 and ATG18) are conserved in mammals, which indicates that
autophagy is an evolutionarily conserved process. As in yeast, mamma-
lian core ATG proteins can be classified into several functional units:
the ULK complex, ATG9L, the class III PI(3)K (phosphatidylinositol-
3-OH kinase) complex, the ATG2-WIPI complex, the ATG12 conjuga-
tion system, and the LC3 conjugation system (Fig. 1).
The ULK complex (a serine-threonine kinase complex composed
of ULK1, ULK2, ATG13, ATG101 and FIP200) and ATG9L (ATG9L1
and ATG9L2) are thought to be the most upstream components in
the autophagy pathway5. The activity of the ULK complex is regu-
lated negatively by the kinase complex mTORC1 and is regulated
positively by the kinase AMPK. Both the ULK complex and ATG9L1

it has also become clear that proteins involved in autophagy have func-
tions other than autophagy as well. In this Review, we will discuss
advances in the understanding of autophagy and its machinery as it
is related to innate immunity, inflammatory responses and adaptive
immunity.
Autophagy proteins and their function
Although autophagic structures were reported in electron microscopy
studies as early as the 1950s, the molecular mechanism of autophagy
remained mostly unknown until the discovery of yeast autophagy-
1Laboratory of Intracellular Membrane Dynamics, Graduate School of Frontier
Biosciences, Osaka University, Osaka, Japan. 2Department of Genetics, Graduate
School of Medicine, Osaka University, Osaka, Japan. 3Division of Molecular
Genetics, Institute for Enzyme Research, Tokushima University, Tokushima,
Japan. 4Viral Immunobiology, Institute of Experimental Immunology, University of
Zrich, Zrich, Switzerland. 5Present address: Laboratory of Veterinary Hygiene,
Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi Japan.
Correspondence should be addressed to T.Y. (tamyoshi@fbs.osaka-u.ac.jp).
Received 11 June; accepted 17 August; published online 18 September 2015;
doi:10.1038/ni.3273.
are required for recruitment of the autophagy-specific class III PI(3)
K complex5,6.
PI(3)Ks are lipid kinases that phosphorylate phosphatidylinositol
at the hydroxyl group at position 3 of the inositol ring, which results
in the generation of phosphatidylinositol 3-phosphate. The autoph-
agy-specific PI(3)K complex (called the ATG14L complex here) is
composed of ATG14L, VPS34 (the catalytic subunit), beclin-1 and
VPS15. ATG14L localizes on the endoplasmic reticulum (ER), and
this localization is required for its autophagy-inducing ability7. The
ATG14L complex accumulates at ER-mitochondria contact sites dur-
ing starvation; this accumulation requires syntaxin-17, a SNARE (sol-
uble N-ethylmaleimide-sensitive factorattachment protein receptor)
protein that resides on the ER and mitochondria8. It is likely that the
ATG14L complex generates phosphatidylinositol 3-phosphate on the
ER at the ER-mitochondria contact site to generate a platform for the
formation of an isolation membrane. The generation of phosphati-
dylinositol 3-phosphate provides the binding site for its binding pro-
teins, including members of the WIPI family (WIPI1WIPI4), the
mammalian orthologs of yeast ATG18. These binding proteins form
a complex with ATG2 (ATG2A and ATG2B). A study has shown that
WIPI2 directly binds to ATG16L1 and recruits the ATG16L1 complex
to the autophagosome-formation site9.

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 REVIEW

Figure 1 The autophagy pathway. Autophagy-

Amino acid starvation
inducing signals (for example, amino acid Pathogen infection, etc.
starvation and pathogen infection) initiate Cell membrane
formation of the isolation membrane (or
phagophore). Closure of the isolation
membrane results in formation of the
autophagosome. The subsequent lysosome- ULK FIP200
autophagosome fusion leads to degradation of ATG13 ATG101 Isolation membrane Autophagosome Autolysosome
the contents of the autophagosome by lysosomal ULK1 complex
hydrolases in the autolysosome. Labels in green
boxes indicate core ATG proteins required for

Kim Caesar/Nature Publishing Group

autophagosome formation.
ATG9L

The ATG16L1 complex (composed of

ATG16L1, ATG5 and ATG12) specifically local- ATG14L VPS34
izes to the isolation membrane and dissociates Beclin-1 VPS15 LC3 Fusion
from it upon the completion of autophagosome ATG14L complex WIPI ATG16L1 ATG5 ATG12 LC3
Hydrolases
formation10. The ubiquitin-like protein ATG12 ATG2 ATG16L1 complex
Lysosome
is covalently conjugated to ATG5 by ATG7
(an E1-like enzyme) and ATG10 (a ubiquitin
E2-like enzyme)11. ATG16L1 and ATG12-ATG5 form a 2:2:2 complex12. including ligands for pattern-recognition receptors (PRRs) (for
This ATG16L1 complex functions as the E3-like enzyme for the other example, Toll-like receptors (TLRs) and Nod-like receptors (NLRs))
ubiquitin-like system: LC3-PE11. There are seven mammalian orthologs and cytokines (Box 2). In addition, the stimulation of phagocytes by
2015 Nature America, Inc. All rights reserved.

of ATG8 (LC3A, LC3B, LC3C, GABARAP, GABARAPL1, GABARAPL2
and GABARAPL3; these are represented by LC3 in this Review). LC3 is
conjugated to the membrane lipid molecule phosphatidylethanolamine
(PE) by ATG7 (an E1-like enzyme) and ATG3 (an E2-like enzyme)11.
LC3-PE conjugation takes place at the isolation membrane, where the
ATG16L1 complex (an E3-like enzyme) accumulates13. LC3 is widely
used as a marker for the microscopic detection of isolation membranes
and autophagosomes. In addition, PE-conjugated LC3 (LC3-II) and
unconjugated LC3 (LC3-I) can be detected separately by immunoblot
analysis, and the amount of LC3-II is also widely used for the quantifica-
tion of autophagic activity14. It has been suggested that LC3 functions in
the closure of the isolation membrane15,16.
Autophagy and pathogen infection
Autophagy has been studied in relation to infection by bacteria,
viruses, and parasites. Various in vivo studies have shown that autoph-
agy serves a protective function against pathogens (Box 1). Other stud-
ies have suggested that autophagy suppresses excessive inflammatory
npg
particles coated with TLR ligands also induces LC3-PE conjugation on
phagosomal membranes. This phenomenon is called LC3-associated
phagocytosis (LAP) (Fig. 2). We note that phagosomes (which have a
single membrane) involved in LAP are different from autophagosomes
(which have a double membrane) and that the detection of LC3 by
fluorescence microscopy should be interpreted carefully, because this
technique cannot distinguish these two structures. Although LAP was
initially shown to facilitate phagosome-lysosome fusion and accelerate
pathogen killing17, further investigation has revealed various functions
for LAP beyond the acceleration of pathogen killing, including antigen
presentation (discussed below in the section on adaptive immunity).
Xenophagy
Although the role of autophagy and ATG proteins in the protection
from pathogen infection in vivo is probably complex, the most direct
method of autophagy-dependent elimination of pathogens is engulf-
ment of the pathogen by the autophagosome and subsequent killing
of the pathogen by lysosome-autophagosome fusion.

responses to pathogen infection. The relationship between autophagy
and inflammation will be discussed later in this Review.
Regulation of the autophagy machinery by immunological signals
Autophagy is regulated by a plethora of immunological signals,
Autophagy was initially thought to be a nonspecific degradation
process. However, it is now apparent that autophagy has an ability
to selectively target large structures, such as intracellular pathogens,
damaged organelles and protein aggregates. This type of autophagy is
called selective autophagy and is thought to maintain intracellular

Box 1 In vivo roles of autophagy in pathogen infection

In mice, knockout of genes encoding autophagy proteins must be conditional, since systemic knockout of these genes results in
death shortly after birth1. For example, knockout of Atg5 in myeloid cells resulted in diminished resistance to L. monocytogenes and
Toxoplasma gondii50 and M. tuberculosis80,91. Knockout of Atg5 or Atg16l1 in intestinal epithelial cells also results in diminished
resistance to Salmonella ser. Typhimurium149,150. The survival of neonatal mice after infection with Sindbis virus is diminished by
neuron-specific knockout of Atg5 (ref. 42). Knockout of Atg5 in DCs diminishes the survival of mice after infection with herpes simplex
virus type 2 (ref. 108). In Drosophila, the knockdown of ATG proteins diminishes host survival after infection with L. monocytogenes151.
Autophagy suppresses the replication of vesicular stomatitis virus in Drosophila152. Caenorhabditis elegans treated by RNA-mediated
interference targeting genes encoding ATG proteins, as well as Dictyostelium expressing mutant ATG proteins, showed reduced survival
after infection with Salmonella ser. Typhimurium153.
In contrast, however, mice with systemic hypomorphic Atg16l1, which show abnormality in the packaging and secretion of
antimicrobial granules by Paneth cells64,154, exhibit greater resistance to urinary tract infection with uropathogenic E. coli and
intestinal infection with Citrobacter rodentium, with enhanced inflammatory cytokine responses and more recruitment of monocytes
and neutrophils to infected sites70,155. The greater clearance of bacteria in these mice might be partly explained by the heightened
inflammatory responses seen in autophagy-deficient macrophages 60.

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Box 2 Immunological signals that regulate autophagy

The stimulation of TLR4 by lipopolysaccharide induces autophagosomes in macrophages156. Other TLR ligands also induce green
fluorescent proteintagged LC3 dots and autolysosomes in macrophages157. Nod1 and Nod2, which are cytosolic receptors for bacterial
peptidoglycan, interact with ATG16L1 and mediate the recruitment of ATG16L1 to the bacterial entry site in non-phagocytic cells,
macrophages and lymphocytes158. Stimulation of Nod2 also induces autophagosomes in DCs159. NLRX1 is required for vesicular
stomatitis virusinduced autophagy by interacting with the ATG16L1 complex through the mitochondrial protein TUFM 87. In contrast,
other NLRs, including NLRC4, NLRP3, NLRP4 and NLRP10, suppress autophagy by sequestering beclin-1 (ref. 160). The alarmin
HMGB1, which is released into the extracellular space and activates certain TLRs, is required for autophagy and the suppression of
endotoxin-induced death of macrophages161. HMGB1 has also been shown to bind to beclin-1 and induces autophagy by releasing
beclin-1 from the antiapoptotic protein Bcl-2 (ref. 162). HIV-1 envelope glycoproteins expressed at the cell surface activate autophagy and
cause apoptosis in bystander CD4+ T cells through the chemokine receptor CXCR4 (ref. 163). IFN-g has been shown to induce autophagy
in macrophages in a manner dependent on the GTPase family member Irgm1 (refs. 21,164). TNF induces autophagy in myoblasts in a
manner independent of the transcription factor NF-kB165 and enhances the recruitment of NDP52 to Shigella species in HeLa human
cervical cancer cells166. IL-1b also induces autophagy in macrophages in a manner dependent on the adaptor TBK-1 (ref. 167). In
contrast, the type 2 helper T cell cytokines IL-4 and IL-13 suppress autophagy168. The transcription factor STAT3, which is activated by
various cytokines, has been shown to suppress autophagy through inhibition of protein kinase R 169.

homeostasis by eliminating unwanted structures. In particular, selec- E3 ubiquitin ligases recognize substrates and therefore determine
tive autophagy directed against pathogens is called xenophagy. substrate specificity. Ubiquitination has been well characterized as a
Antibacterial xenophagy in both non-phagocytic cells and phago- hallmark event in the ubiquitin-proteasome pathway, which degrades
2015 Nature America, Inc. All rights reserved.

cytic cells has been investigated extensively. For phagocytes such as
macrophages, dendritic cells (DCs) and neutrophils, bacteria are rec-
ognized by PRRs on the phagocyte surface, which leads to the activa-
tion of intracellular signaling that induces immune responses. At the
same time, bacteria are internalized by phagocytosis and are effectively
killed by phagosome-lysosome fusion as well as by exposure to reactive
oxygen species (ROS) and antimicrobial peptides18. However, inva-
sive bacteria, such as Salmonella, Listeria and Shigella species, invade
non-phagocytic cells (for example, cells of the intestinal epithelium).
They modify endosomes into a niche suitable for their survival and
proliferation and often break into the nutrient-rich cytosol from the
vacuole19.
Xenophagy directed against invasive bacteria in non-phagocytic
cells was first demonstrated in a study showing that the bacterium
group A Streptococcus (GAS) is efficiently targeted by an LC3+
autophagosome-like double-membraned structure and is later sub-
jected to lysosomal degradation20. Moreover, this study showed that
genetic deletion of Atg5 in mice results in the absence of the LC3+
npg
individual ubiquitinated proteins by the proteasome26. However, stud-
ies have also shown its involvement in selective autophagy. Notably,
the fusion of ubiquitin to monomeric red fluorescent protein or
peroxisomes leads to autophagic degradation of the monomeric red
fluorescent protein or peroxisomes, respectively27. Moreover, invasive
bacteria often localize together with ubiquitin28. Two E3 ubiquitin
ligases have been related to antibacterial xenophagy thus far: LRSAM1
(refs. 29,30) and parkin31. Parkin is also known as the main E3 ubiq-
uitin ligase responsible for mitophagy (the selective autophagy of
mitochondria)32.
One possible mechanism underlying the bacteria-ubiquitin colo-
calization noted above might be direct ubiquitination of bacteria, and
an alternative possibility is the ubiquitination of bacteria-containing
endosomes. Several studies support the latter model. The endosomal
membrane is ubiquitinated after S. flexneri escapes into the cytosol
from the endosome33. Salmonella ser. Typhimurium inside endosomes
has been found to be targeted by autophagosomes6,23. Strikingly, even
latex beads ingested by endocytosis can induce autophagosome forma-

structure and in improved survival of GAS. This study clearly showed
that autophagy functions as an intracellular innate immune system
directed against bacterial invasion in non-phagocytic cells. Around
the same time as that study, another study showed that the survival of
Mycobacterium bovis bacillus Calmette-Gurin and Mycobacterium
tuberculosis in macrophages is suppressed by the induction of autoph-
agy21. Follow-up studies have shown that other invasive bacteria,
including Shigella flexneri22, Salmonella enterica serovar Typhimurium
(Salmonella ser. Typhimurium)23 and Listeria monocytogenes24, are
also targeted by autophagy.
However, there are bacterial species that have evolved mechanisms
to prevent autophagy in a strain-specific manner and therefore have
become more vulnerable to antibacterial xenophagy when these self-
defense mechanisms are impaired. These bacteria include S. flexneri,
L. monocytogenes and Legionella pneumophila, as reviewed in detail
elsewhere25.
Mechanisms of xenophagy: ubiquitination of the substrate
The key event that triggers antibacterial xenophagy and other selective
autophagy is ubiquitination (the covalent conjugation of ubiquitin to
a substrate). Ubiquitination is mediated by E1 (ubiquitin-activating
enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin ligase).
tion after the bead-containing endosomal membrane is ruptured34,35,
which suggests that no bacterial factors are required for the selective
formation of autophagosomes. Moreover, rupture of the lysosomal
membrane induces selective ubiquitination and autophagy of the
ruptured lysosome (a process called lysophagy)36,37. An important
insight from these results is that cells might be simply detecting the
rupture in pathogen-containing endosomes as an autophagy-inducing
signal rather than preparing different autophagy-inducing mecha-
nisms for various kinds of pathogens.
Recruitment of LC3 through autophagy adaptors
How can ubiquitination function as the signal for selective autophagy?
Autophagy adaptors (receptors) such as the ubiquitin sensor SQSTM1
(p62), NDP52 and optineurin have been suggested to serve an impor-
tant role in xenophagy directed against intracellular bacteria25. These
adaptors contain ubiquitin-binding domains and LC3-interacting
regions, and therefore it is proposed that these adaptors recruit the LC3
machinery to ubiquitinated substrates (Fig. 2). In addition, NDP52
binds galectin-8, a b-galactose-binding lectin. Since b-galactose chains
exist in the lumen of endosomes, galectin-8 and its binding partner
NDP52 are recruited from the cytoplasm to the endosomal membrane
when the endosome is ruptured38.

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Figure 2 Xenophagy and LAP. In ubiquitin-

dependent xenophagy (left), the recruitment
of ATG proteins occurs after the pathogen- Ubiquitin-dependent
containing endosome is ruptured. Endosomal xenophagy LAP in phagocytes
membrane proteins are ubiquitinated (Ub) Pathogen
immediately after the membrane rupture. In (bacteria, etc.)
addition, adaptors such as SQSTM1 (p62), Cell membrane
NDP52 and optineurin mediate recruitment
of LC3 to the ubiquitinated substrate. NDP52
can be recruited to the ruptured endosomal
membrane through binding to galectin-8, which
recognizes b-galactose chains in the lumen of
endosomes (not shown here). Formation of the
isolation membrane takes place in the proximity
of the pathogen-containing endosome, and
finally, the autophagosome engulfs the pathogen-
containing endosome. The subsequent lysosome-
autophagosome fusion eliminates the pathogen.
In contrast to the process used for xenophagy,
the double-membraned autophagosome is not WIPI or
formed in LAP (right). Instead, the phagosomal ATG2

membrane is decorated directly by LC3. LAP Ub ULK

complex
facilitates lysosome-phagosome fusion or ATG14L1
complex
prolonged antigen processing for presentation by
2015 Nature America, Inc. All rights reserved.

Ub ATG9L LC3
MHC class II in a cell typespecific manner.
ATG16L1
Ub complex LC3

Recruitment of ATG proteins

Ub Adaptor LC3
However, even in the absence of the recruit- Fusion
ment of LC3 to intracellular Salmonella
species (in cells deficient in Atg3, Atg5 or
Atg7), formation of an isolation membrane Lysosome or
can initiate around Salmonella-containing endosome

Kim Caesar/Nature Publishing Group

endosomes6. In the absence of LC3 recruit-
ment, other ATG proteins such as the ULK1
complex and ATG9L1 are recruited to intra-
Fusion
cellular Salmonella, which suggests that the
recruitment of these early ATG proteins is
sufficient to begin the formation of an isola- Lysosome
tion membrane independently of the adap-
tor-mediated recruitment of LC36, although
these isolation membranes cannot mature genes, SMURF1, has been shown to be required for cell survival after
to complete autophagosome formation without LC3-PE. A follow-up infection with Sindbis virus or herpes simplex virus type 1 (ref. 43).
npg

study has revealed that recruitment of the ULK1 complex, ATG9L1
and the ATG16L1 complex (and the downstream ATG proteins
ATG14L, WIPI1 and LC3) to ruptured endosomes is all dependent
on ubiquitination35. Recruitment of the ATG16L1 complex involves
direct ATG16L1-ubiquitin binding through the carboxy-terminal WD
domain of ATG16L1 and direct ATG16L1-FIP200 binding (FIP200
is a subunit of the ULK1 complex)35. The exact mechanisms of the
ubiquitination-dependent recruitment of the ULK1 complex and
ATG9L1 remain unknown.
Xenophagy directed against viruses
The role of autophagy in viral infection is complex and depends on the
virus. Although several viruses have autophagy-inhibitory functions
and even exploit autophagy for their replication39,40, the virulence of
several other viruses is suppressed by autophagy. For example, capsid
proteins of Sindbis virus are degraded by autophagy in an SQSTM1-
dependent manner, and mice with neuron-specific Atg5 deficiency
exhibit greater susceptibility to infection with Sindbis virus than do
their wild-type counterparts41,42. Genome-wide screening with small
interfering RNA has identified 141 genes whose knockdown results
in a change in the colocalization of Sindbis virus capsid protein with
green fluorescent proteintagged LC3. The product of one of those
The human immunodeficiency virus type 1 (HIV-1) virulence fac-
tor Nef has been shown to suppress autophagy by binding to beclin-1
(ref. 44). The Nef-binding sequence of beclin-1 (amino acids 267284)
induces autophagy when modified as a cell-permeable peptide (Tat
beclin-1). Tatbeclin-1 suppresses Sindbis virus, Chikungunya virus,
West Nile virus and HIV-1 in vitro and diminishes the lethality of
infection with Chikungunya virus or West Nile virus in vivo45.
Autophagy-independent functions of ATG proteins
The role of ATG proteins in immunity is not restricted to xenophagy.
They can also function in autophagy-independent ways. One striking
example is LAP. Unlike the process of canonical autophagy, single-
membrane phagosomes are modified by LC3 during LAP in a way
dependent on ATG5, ATG7 and beclin-1 but independent of ATG14L
or the ULK1 complex17,4648. In another example, Brucella abortus
exploits autophagic machinery by forming autophagic Brucella-
containing vacuoles. Although these vacuoles have double-mem-
braned structures reminiscent of autophagosomes and require ULK1,
beclin-1 and ATG14L, they lack the autophagosome marker LC3 and
do not require ATG5 or ATG16L1 (ref. 49). Other examples of autoph-
agy-independent functions of ATG proteins include ATG5-dependent
membrane damage in Toxoplasma-containing vesicles50, ATG16L1

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 REVIEW
complexdependent inhibition of the formation of membranous rep-
lication complex in mouse norovirus infection51, and LC3-associated
ejection of intracellular pathogens such as poliovirus52, coxsackie
virus53 and uropathogenic Escherichia coli54 toward the extracellular
space. Other examples are also discussed below. Such examples indi-
cate that the observation of one feature associated with autophagy (for
example, the recruitment of LC3, the formation of double-membrane
structures, etc.) does not necessarily guarantee that the phenomenon
is canonical autophagy. Therefore, such phenomena should be inter-
preted carefully, and the involvement of autophagy should be assessed
by multiple, functionally different experiments.
Autophagy in innate immunity
The innate immune system induces inflammation to protect the host
from microbial infection55. PRRs sense microbial components and
induce activation of signal-transduction pathways, which leads to the
expression of inflammatory mediators such as cytokines and type I
interferons. Although inflammation is essential for host defense, aber-
rant induction of inflammation causes the development of inflam-
matory diseases, such as septic shock, autoimmune diseases and
metabolic diseases. Thus, the innate immune system is strictly regu-
2015 Nature America, Inc. All rights reserved.
lated to prevent either insufficient inflammatory responses or excessive
inflammatory responses. Accumulating evidence has shown that the
intracellular degradation system has an important role in regulating
inflammatory responses. In particular, studies have shown the involve-
ment of autophagy in inflammatory responses56.
Autophagy in inflammasome activation
The inflammasome is a cytosolic protein complex consisting of NLRs
or of the cytosolic receptor AIM2 together with the adaptor ASC
and the protease caspase-1 (ref. 57). Activation of inflammasomes
induces the proteolytic maturation of interleukin 1b (IL-1b) and the
related cytokine IL-18 by caspase-1, which in turn induces produc-
tion of these cytokines. Inflammasomes drive cytokine-dependent
host defense against microbes. However, aberrant activation of
inflammasomes by an excess of microbial components, host-derived
stimulatory factors and environmental irritants often causes massive
inflammation, which results in severe tissue damage. Hence, inflam-
masomes are closely associated with the development of infectious
npg
and inflammatory diseases.
Genome-wide association studies have identified ATG16L1 as a gene
encoding a factor responsible for susceptibility to Crohns disease, a
chronic inflammatory disease of the intestine58,59. The importance
of ATG16L1 in the maintenance of intestinal homeostasis has been
confirmed in mouse models. Mice with hematopoietic cellspecific
deficiency in ATG16L1 and mice that express the Crohns disease
associated ATG16L1 variant ATG16L1 T300A are susceptible to colitis
induced by dextran sodium sulfate and colitis induced by pathogenic
bacteria, respectively6062. ATG16L1-deficient macrophages produce
large amounts of IL-1b and IL-18 in response to lipopolysaccharide,
ATP or monosodium urate crystals (Fig. 3). Expression of the Crohns
disease-associated variant ATG16L1 T300A in peripheral blood mono-
nuclear cells from patients with Crohns disease and in macrophages
from genetically engineered mice results in elevated production of
IL-1b6163. Caspase-3 and caspase-7, which are apoptotic proteases,
enhance the production of IL-1b by inducing proteolytic cleavage of
the Crohns diseaseassociated variant ATG16L1 T300A61,62. Loss
of autophagy in macrophages greatly augments the production of
IL-1b, as macrophages deficient in ATG7 or treated with an inhibi-
tor of the ATG14L complex catalytic subunit VPS34 produce large
amounts of IL-1b in response to various inflammasome inducers60.
1018 VOLUME 16 NUMBER 10 OCTOBER 2015 NATURE IMMUNOLOGY
However, ATG16L1 in intestinal epithelial cells also controls inflam-
mation. Hypomorphic mutation of Atg16l1 in mice results in Paneth
cell abnormality due to infection with norovirus and induces elevated
production of tumor-necrosis factor and interferon-g (IFN-g), which
results in enhanced susceptibility to dextran sodium sulfateinduced
colitis64. Hence, autophagy has a key role in the control of inflamma-
tory cytokine production.
NLRP3 is a well-characterized PRR of the NLR family that induces
the production of IL-1b and IL-18 (ref. 57). Various inducers of the
NLRP3 inflammasome, such as ATP, palmitic acid, monosodium urate
crystals, silica nanoparticles, cholesterol crystals and influenza A virus,
cause mitochondrial damage, which leads to the release of ROS. NLRP3
forms an inflammasome complex with ASC and caspase-1 and induces
the production of IL-1b and IL-18 in response to mitochondrial ROS.
Because autophagy maintains cellular homeostasis by promoting the
turnover of organelles, a deficiency in autophagy results in the accu-
mulation of old and dysfunctional mitochondria. Accumulated old and
dysfunctional mitochondria produce an excess of ROS upon stimu-
lation, which potently activates the NLRP3 inflammasome65,66 (Fig.
3). In addition, lysophagy can also be important for suppressing the
activation of inflammasomes, since extracellular irritant particles such
as monosodium urate and silica are internalized into cells via the endo-
cytic pathway and break lysosomes, which might damage mitochon-
dria. Lysophagy sequesters lysosomes damaged by monosodium urate
or silica in vitro and in vivo37. Upon infection with influenza A virus,
the cytosolic PRR Nod2 and its downstream regulator RIPK2 induce
mitophagy by inducing phosphorylation of ULK1 to prevent excessive
activation of the NLRP3 inflammasome67. A deficiency in autophagy
worsens the symptoms of various inflammatory conditions, such as
colitis, sepsis, pneumonia, diabetes, atherosclerosis and urinary tract
infection, by enhancing NLRP3 inflammasomedependent production
of IL-1b and IL-18 (refs. 6670). In contrast, inducers of autophagy
can suppress the inflammasome-dependent production of IL-1b and
IL-18 and ameliorate symptoms of inflammation-associated tissue
damage7173. Hence, autophagy might be a useful therapeutic target
for the treatment of inflammasome-related inflammatory diseases.
Autophagy inhibits not only the NLRP3 inflammasome but also
other inflammasomes, such as the AIM2 inflammasome. Lys63
(K63)-linked ubiquitination of ASC, an essential adaptor of inflam-
masomes, is induced upon activation of the NLRP3 and AIM2 inflam-
masomes74. Autophagy recognizes ubiquitinated ASC by the ubiquitin
sensor SQSTM1 and induces selective degradation of inflammasomes
and thereby suppresses the production of IL-1b and IL-18 (Fig. 3).
Furthermore, the autophagy system delivers IL-1b into lysosomal com-
partments to induce its degradation75 (Fig. 3). Hence, autophagy can
target multiple steps of inflammasome activation and thus can potently
inhibit the production of IL-1b and IL-18.
Autophagy limits the activation of inflammasomes under normal
and nutrient-rich conditions. However, a study has shown that autoph-
agy often mediates unconventional release of IL-1b under conditions
of nutrient starvation76. GORASP and RAB8A, which are membrane
traffic regulators, are involved in the autophagy-dependent uncon-
ventional release of IL-1b. However, other studies have shown that
autophagy suppresses IL-1b production under conditions of nutri-
ent starvation70,74. Hence, the role of autophagy in IL-1b production
under conditions of nutrient starvation is still unclear and needs to
be investigated further.
Other studies have shown that the non-canonical inflammasome
consisting of caspase-11 in mice and caspase-4 and caspase-5 in
humans induces the production of IL-1a and/or IL-1b and IL-18 in
response to cytosolic lipopolysaccharide77. During infection with
 REVIEW

DNA virus Bacteria Uric acid crystals Influenza Palmitic LPS ATP
Cholesterol crystals virus acid

TLR4 P2X7 Cell membrane

Phagosome
damage

Phagosome Autophagy
damage induction

Autophagosomes

dsDNA LPS
leakage leakage
Non- Mitochondria
canonical damage
pathway ROS Engulfment
generation of substrates
AIM2 NLRP3 Ub
PRR activation Ub Ub
2015 Nature America, Inc. All rights reserved.

ASC

Ub Lysosome
ASC oligomerization ASC Ub Ub
fusion

Cytokine production IL-1

Kim Caesar/Nature Publishing Group

Autophagic
degradation
Inflammation

Autolysosomes

Figure 3 Autophagy suppresses the inflammasome-mediated production of IL-1b. NLRP3 forms an inflammasome with its adaptor ASC, and its excessive
activation causes inflammatory diseases such as gout. Activators of the NLRP3 inflammasome cause mitochondrial damage, which in turn causes the
production of ROS; this results in activation of the NLRP3 inflammasome. Autophagy induces the elimination of old and dysfunctional mitochondria to
suppress ROS generation and subsequent activation of the NLRP3 inflammasome. Autophagy also induces the elimination of damaged phagosomes,
npg

ubiquitinated ASC and IL-1b to suppress IL-1b production mediated by other inflammasomes. LPS, lipopolysaccharide.

Gram-negative bacteria, a cluster of guanylate-binding proteins,
which are small interferon-inducible GTPases, moves to the surface
of the bacteria-containing vacuoles (BCVs) and induces membrane
rupture78. The destabilization of BCVs releases bacterial components
into the cytosol and thereby facilitates recognition of their lipopolysac-
charide by the non-canonical inflammasome. A deficiency in autoph-
agy causes accumulation of the destabilized BCVs, which results in
enhanced activation of the non-canonical inflammasome (Fig. 3).
Autophagy targets damaged BCVs via ubiquitin and galectin-8 and
induces their autophagic degradation as described above. Hence,
autophagy-mediated clearance of BCVs prevents excessive induction
of the inflammatory response mediated by the non-canonical inflam-
masome.
Autophagy in inflammasome-independent IL-1 production
The maturation and production of IL-1a involve caspase 1-depen-
dent and caspase 1-independent processes79. In caspase-1-indepen-
dent process, the maturation of IL-1a by calpain is triggered by ROS
released from damaged mitochondria. A deficiency in autophagy in
phagocytes causes enhanced responses of the TH17 subset of helper
T cells, which results in severe lung tissue damage in a mouse model
of tuberculosis80. Autophagy-deficient macrophages produce large
amounts of IL-1a in response to M. tuberculosis due to an elevation
in mitochondrial ROS. Hence, mitochondrial ROS have a critical role
in activation of the inflammasome and calpain in autophagy-deficient
macrophages.
Autophagy in type I interferon production
The innate immune system senses viral nucleic acids through PRRs
such as TLRs, RLRs and cGAS and induces production of the type
I interferons IFN-a and IFN-b8183. Type I interferons induce the
expression of a series of antiviral factors to establish an antiviral state
and have a crucial role in host defense against viruses.
TLR7 and TLR9 sense viral single-stranded RNA and unmethylated
CpG DNA, respectively, in endolysosomes and mediate robust produc-
tion of type I interferons by plasmacytoid DCs (pDCs)81. Loss of ATG5
severely impairs TLR7-dependent production of type I interferons by
pDCs infected with vesicular stomatitis virus or Sendai virus84. Loss
of ATG5 also severely impairs TLR9-dependent production of type I
interferons by pDCs stimulated with A- or D-type unmethylated CpG

NATURE IMMUNOLOGY VOLUME 16 NUMBER 10 OCTOBER 2015 1019

 REVIEW
oligonucleotides (ligands for TLR9). Furthermore, LAP deficiency
severely impairs pDC production of type I interferon induced by com-
plexes of DNA and immunoglobulins and dependent on TLR9 (ref.
48). Hence, autophagy and LAP are required for the sensing of nucleic
acids and TLR-mediated production of type I interferons by pDCs.
The RLRs RIG-I and Mda5 are caspase-recruitment domaincon-
taining RNA helicases and sense viral double-stranded RNA (dsRNA)
in the cytosol82. After sensing viral dsRNA, RLRs induce the produc-
tion of type I interferons through activation of the mitochondrial
caspase-recruitment domaincontaining adaptor IPS-1 (also known
as MAVS, VISA and CARDIF). ATG5 deficiency enhances the produc-
tion of type I interferons by RNA viruses such as vesicular stomatitis
virus and inhibits viral replication. ATG5 deficiency results in the
accumulation of old and dysfunctional mitochondria, which in turn
induces elevated expression of IPS-1 and the generation of mitochon-
drial ROS; this results in enhanced production of type I interferons by
dsRNA85. ATG12-ATG5 conjugates and their binding partner TUFM
suppress dsRNA-induced production of type I interferons by disrupt-
ing signals from RLRs and IPS-1 (refs. 86,87). Hence, autophagy and
a unique function of autophagy-related proteins are involved in nega-
tive regulation of the RLR-mediated production of type I interferons.
2015 Nature America, Inc. All rights reserved.
Studies have shown that cGAS senses microbial dsDNA in the cyto-
sol83. After recognizing dsDNA, cGAS generates cGAMP to activate
STING, a downstream regulator, which in turn induces the production
of type I interferons; this results in the establishment of an antiviral
state. Loss of ATG9L1 causes hyper-aggregation of STING on Golgi-
derived compartments and enhances STING-dependent production
of type I interferons88. ATG9L1 controls the subcellular localiza-
tion of STING via unknown mechanisms. Inhibition of ULK1 also
enhances the STING-dependent production of type I interferons89.
ULK1 induces the phosphorylation of STING to prevent sustained
production of type I interferons. Inhibition of beclin-1 enhances
cGAS-dependent production of type I interferons. Beclin-1 binds to
cGAS to inhibit cGAMP synthesis90. In contrast, the cGAS-STING
signaling axis is required for the targeting of autophagosomes to and
their elimination of M. tuberculosis9193. Hence, both autophagy and
unique functions of autophagy-related proteins regulate the cGAS-
mediated anti-microbial response.
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Endogenous antigen processing by autophagy
In contrast to sensing by the innate immune system via PRRs, the adap-
tive immune system relies on the recognition of peptides processed
from pathogen- or tumor-associated proteins and their presentation on
major histocompatibility complex (MHC) molecules. For this purpose,
MHC class I molecules are loaded mainly with proteasomal products
for their recognition by CD8+ T cells, while MHC class II molecules
receive fragments of lysosomal degradation for their recognition by
CD4+ T cells94. Autophagy delivers cytoplasmic constituents for lyso-
somal hydrolysis and thereby contributes to the processing of endog-
enous antigens for presentation by MHC class II molecules95 (Fig. 4).
This was first noted for a prominent antigen of Epstein-Barr virus
recognized by CD4+ T cells96,97. Further studies have demonstrated
that rapamycin-induced autophagy or targeting of model antigens to
autophagosomes by fusion to LC3 augments the presentation of intra-
cellular antigens on MHC class II molecules98100. However, the best-
characterized physiological process that requires intracellular antigen
loading is T cell education in the thymus101. Thymic epithelial cells
present self antigens on MHC molecules for the induction of central T
cell tolerance. Indeed, thymic epithelial cells have high constitutively
active levels of autophagy101,102; absence of this autophagy results in
the compromised education of CD4+ T cells101. The targeting of anti-
1020 VOLUME 16 NUMBER 10 OCTOBER 2015 NATURE IMMUNOLOGY
gens to the autophagy machinery results in the depletion of specific T
cells during negative selection, which eliminates autoreactive T cells103.
Therefore, the processing of endogenous MHC class II antigens via
autophagy contributes to the induction of T cell tolerance and pro-
motes the presentation of intracellular antigens to CD4+ T cells.
Exogenous antigen processing for presentation by MHC class II
In addition to the function noted above of classical autophagy in anti-
gen processing, the LC3-PEconjugation machinery also regulates
the processing of exogenous antigens via phagocytosis. During this
LAP, LC3 is coupled to a subset of phagosomal membranes that sur-
round TLR2, TIM4, Fc receptors or dectin-1-binding cargo17,46,104106.
The production of ROS seems to be needed to maintain the conjuga-
tion of LC3 to phagosomes involved in LAP47,105. The fate of these
phagosomes depends on the cellular background in which they form.
Mouse macrophages rapidly degrade the contents of LAP phago-
somes in lysosomes, possibly by LC3B-dependent recruitment of
the vesicular transport protein FYCO1, which might accelerate the
microtubule transport of LAP phagosomes17,46,107. In contrast, pDCs
and human macrophages, as well as monocyte-derived DCs, seem to
stabilize these vesicles for fusion with TLR-containing endosomes and
prolonged processing of antigen for presentation by MHC class II,
respectively48,105. Cargo that engages dectin-1 or TLR2 seems to be
efficiently processed via LAP for presentation by MHC class II mol-
ecules105,106 (Fig. 4). Similarly, the LC3-PEconjugation machinery
seems to be required for efficient presentation by MHC class II in
vivo108. Moreover, hydrolases that facilitate antigen processing can be
transported into lysosomes via autophagy, as shown for peptidylargi-
nine deiminases that generate citrullinated peptides, which constitute
epitopes that are recognized by CD4+ T cells in rheumatoid arthri-
tis109. However, LAP does not seem to affect all phagocytosed cargo
similarly. Although the autophagy machinery has been reported to
assist the transport of antigens recognized by B cell antigen receptors
to TLR-containing endosomes110, this does not seem to be required
for the establishment of the presentation of antigens to helper T cells
during germinal center reactions111. Thus, the autophagy machinery
that mediates LC3-PEconjugation can modify the fate and content
of phagosomes and facilitate the presentation of exogenous antigens
by MHC class II molecules.
Antigen processing for presentation by MHC class I
The classical function of the LC3-PEconjugation machinery (deliv-
ering substrates for lysosomal degradation) might limit the access of
antigens to proteasomal turnover112. In addition, proteasomes can be
targeted by the autophagy machinery113,114, but this might affect mainly
dysfunctional proteasomes115 and contribute little to further constrain
proteasomal processing of antigens for presentation by MHC class
I molecules112. However, viral infection can block this proteasomal
degradation and the import of antigenic peptides into the ER, medi-
ated by the transporter TAP, for loading onto MHC class I molecules.
Under these conditions, autophagy and possibly even LAP might con-
tribute to the loading of antigen onto MHC class I molecules (Fig. 4).
Indeed, involvement of autophagy in the presentation of antigens by
MHC class I molecules during herpesvirus infection in vitro has been
shown for herpes simplex virus type 1 and human cytomegalovirus,
which both encode TAP-blocking proteins116,117. Notably, autophagy
is reported to assist in the upregulation of MHC class I mediated by
IFN-g118. Therefore, autophagy might contribute to the delivery of
intracellular antigens to vesicular MHC class Iloading compart-
ments and to the processing of extracellular antigens for presenta-
tion by MHC class I, called cross-presentation. Cross-presentation of

Figure 4 ATG proteins in MHC-restricted
antigen presentation. Autophagy-related ATG8 and LC3
proteins contribute, by at least three pathways, MHCII
to the loading of vesicular antigens onto MHC MHCI
molecules. Autophagosomes can fuse with Antigen
endosomal MHC loading compartments and
deliver cytoplasmic constituents for loading onto
MHC class II (MHCII). Loading of MHC class
I (MHCI) might also occur in these vesicular
compartments, especially under conditions in
which the classical processing of classical MHC
class I antigens, including import into the ER by LAP
TAP, is inhibited. LAP can deliver extracellular
antigens to this loading compartment. LC3
might need to be deconjugated from the outer
autophagosomal and the LAP phagosome
membrane by ATG4 for efficient fusion with the ATG4
endosomal MHC loading compartment. Finally,
signal peptideindependent but autophagy-
dependent exocytosis seems to efficiently
transfer antigens from donor cells to antigen- ATG4
presenting cells, mainly for cross-presentation by
MHC class I molecules.
2015 Nature America, Inc. All rights reserved.
Macroautophagy
antigens from respiratory syncytial virus, HIV, Chlamydia bacterial
species and Aspergillus mold species are reported to benefit from the
LC3-PEconjugation machinery119122, presumably via the fusion of
LAP phagosomes with vesicular MHC class I loading compartments.
Hence, vesicular loading of MHC class I might benefit from autophagy
and LAP, especially when TAP is blocked.
Antigen packaging for cross-presentation
In addition to the catabolic functions of the LC3 lipidation machinery,
alterations that affect completion of the autophagosome-maturation
process might promote exocytosis. This has been demonstrated in
yeast for signal peptideindependent secretion123,124 and has been sug-
gested for the release of, among others, the alarmin HMGB1, IL-1b,
ATP and secretory lysosomes by mammalian cells76,125127. Moreover,
some viruses, such as HIV, polio and Epstein-Barr virus, might uti-
npg
lize exocytosis involving autophagic membranes to exit infected
cells44,128,129. This autophagic exocytosis might also benefit antigen
processing and has been shown to facilitate the cross-presentation of
tumor antigens and antigens from influenza A virus130,131 (Fig. 4).
Accordingly, vesicular fractions generated from antigen-donor cells
under autophagy-stimulating and lysosome-inhibiting conditions,
some of which are called defective ribosomal products in blebs (or
dribbles), are now being explored as antigen formulations for better
cross-presentation132. Thus, autophagic membranes might contribute
to antigen release for efficient cross-presentation.
Autophagy in T cells
Beyond its role in antigen processing, autophagy shapes adaptive immu-
nity through its contribution to the development and effector function
of lymphocytes. While myeloid cells seem able to differentiate in the
absence of autophagy and can even cause severe myeloproliferation,
a deficiency in autophagy in hematopoietic progenitor cells in mice
severely compromises the lymphoid lineage133. Accordingly, thymo-
cyte development upregulates and is dependent on functional autoph-
agy134,135. The number of naive T cells is much lower in the absence
of autophagic clearance of damaged mitochondria (mitophagy)136,137.
Thus, mitophagy seems to be a prerequisite for T cell development.
NATURE IMMUNOLOGY VOLUME 16 NUMBER 10 OCTOBER 2015 1021
REVIEW
Cell
membrane
Antigen-
donor cell
Exocytosis of
Endosomal MHC
loading compartment
LC3-decorated
vesicles
Kim Caesar/Nature Publishing Group
Antigen or
presenting cell
Autophagy also has a crucial role in the function of mature T cells.
Proliferating T cells upregulate autophagy134, and the survival of T cell
during proliferation depends on this134,138. A lack of autophagy leads
to terminally differentiated senescent CD8+ T cells with dysfunctional
mitochondria, increased ROS production and elevated amounts of
p38 (a mitogen-activated protein kinase)139. Indeed, p38 suppresses
autophagy in these cells, and its inhibition restores proliferative func-
tion. Furthermore, the altered cell organelle homeostasis with enlarged
ER structures compromises Ca2+ mobilization after T cell stimula-
tion140. This seems to affect not only effector T cells but also regulatory
T cells141. Moreover, CD4+ T cells defective in autophagy are suscep-
tible to apoptosis after they are activated142. In addition, a deficiency
in autophagy causes impaired survival of memory CD8+ T cells during
infection with lymphocytic choriomeningitis virus, influenza virus
or mouse cytomegalovirus143,144. T cell memory responses are par-
tially restored by the autophagy stimulator spermidine143. Therefore,
autophagy might be required for the population expansion and mem-
ory maintenance of lymphocytes.
Autophagy in the development and function of B cells
The loss of autophagy in developing B cells results in a marginally
decreased total number of cells135,145. However, the number of mouse
B-1a cells is substantially compromised when genes encoding essential
ATG proteins are deficient111,145. This decrease seems to originate
from the autophagy-dependent transition from pre-B cell to pro-B
cell. Therefore, a subset of B cells is dependent on autophagy during
their differentiation.
In line with the dependence of B cell development on functional
autophagy, effector functions of the humoral immune response are
also affected by a deficiency in autophagy. The terminal effector func-
tion resides with plasma cells that continuously generate antibodies.
In addition, memory B cells can be reactivated for antibody produc-
tion upon encountering cognate antigen. Both T celldependent
antibody responses and T cellindependent antibody responses that
lead to plasma cell differentiation require autophagy111,146. Moreover,
the long-term survival of plasma cells in the bone marrow of mice is
diminished by a deficiency in autophagy111. The absence of autophagy
 REVIEW
in plasma cells results in massive enlargement of the ER, which trig-
gers unfolded protein responses during the production of immuno-
globulins. This uncontrolled stress response is thought to induce cell
death111. However, a direct effect of autophagy on antibody production
by plasma cells and multiple myeloma cells has not been shown111,147.
In addition to the maintenance of plasma cells, autophagy is also
required for the survival of memory B cells. Mice with autophagy-
deficient B cells have severely impaired specific secondary antibody
responses to influenza A virus due to compromised maintenance of
memory B cells148. Consequently, humoral immune responses, par-
ticularly the protection due to sustained antibody production and
secondary responses, necessitate autophagy.
Concluding remarks
Since the discovery of the ATG proteins, the molecular mechanism of
autophagy and its relationship with immunity has been investigated
extensively. As noted in this Review, it is now indisputable that autoph-
agy and its machinery have critical roles in immunity, including the
elimination of pathogens, the regulation of inflammatory responses,
and antigen processing and presentation, as well as the development
of cells of the immune system. However, the exact functions of ATG
2015 Nature America, Inc. All rights reserved.
proteins and mechanism of autophagosome formation are still in being
elucidated. Future investigations should clarify the fundamental mech-
anism of autophagy in more detail and will probably reveal unexpected
roles for autophagy in immunity.
ACKNOWLEDGMENTS
Supported by the Ministry of Education, Culture, Sports, Science and Technology
of Japan (S.T.S., T.S. and Y.T.), the Japan Agency for Medical Research and
Development CREST (S.T.S., T.S. and Y.T.), Cancer Research Switzerland (KFS-
3234-08-2013 for H.N. and C.M.), Worldwide Cancer Research (14-1033 for
H.N. and C.M.), the clinical research programs KFSPMS and KFSPHHLD of the
University of Zurich (H.N. and C.M.), the Baugarten Foundation (H.N. and C.M.),
the Sobek Foundation (H.N. and C.M.), Fondation Acteria (H.N. and C.M.), the
Swiss Vaccine Research Institute (H.N. and C.M.) and the Swiss National Science
Foundation (310030_143979 and CRSII3_136241 for H.N. and C.M.).
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html.
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