Relative to other autophagy types, macroautophagy (hereafter referred as autophagy) is most important for maintaining homeostasis because it regulates the turnover and functionality of key cellular organelles (). For instance, tissue damage and stress cause the release of danger-associated molecular patterns (DAMP) that are sensed by resident tissue macrophages, which mount an acute inflammatory response whose goal is to get rid of cell corpses and initiate tissue repair and regeneration (). However, macrophage overactivation can result in hyperplasia and tumor promotion. Therefore, the inflammatory response needs to be properly terminated, a task mediated by autophagy (). DAMP-induced macrophage activation often involves mitochondrial stress, which results in release of mitochondrial signals that affect secretion of inflammasome-dependent cytokines to fight infections and promote tissue repair (). However, uncontrolled macrophage activation results in self-inflicted death, which subsequently triggers extensive neutrophil recruitment, thereby causing severe immunopathologies. This dangerous process is counteracted by mitochondrial autophagy (mitophagy) in macrophages (). In addition, mitophagy in epithelial and mesenchymal cells is important for preventing mitochondrial reactive oxygen species (mtROS)-induced tissue damage and tumor initiation (). Moreover, autophagy is required for optimal induction of protective adaptive immunity (). Hence, in addition to its well-recognized cell-autonomous anti-tumorigenic properties, autophagy, as outlined below, also controls important non-cell-autonomous functions that counteract tumorigenesis, mainly through activation of adaptive immunity and inhibition of chronic inflammation.

Although “autophagy” was described in the late 1950s, its mechanism was elucidated much later using yeast genetics (). Autophagosome formation requires three main steps: initiation, nucleation, and expansion ( Figure 1 ), which have been extensively reviewed elsewhere (). Autophagy is classified as microautophagy, chaperone-mediated autophagy and macroautophagy (). Microautophagy involves invagination of either lysosomal or endosomal membranes, resulting in direct engulfment of cytoplasmic cargo. Chaperone-mediated autophagy entails selective degradation of proteins containing a KFERQ-like motif (). Macroautophagy consists of classic double-membrane autophagosomes that recognize and sequester cellular cargo that has been tagged by autophagy adaptors (e.g., sequestosome 1 [p62/SQSTM1], neighbor of Brca 1 [NBR1] and optineurin) (). Cargo recognition often depends on ubiquitylation, but under certain circumstances, non-ubiquitinated cargo is also cleared by autophagy (). Successfully encapsulated cargo is eventually degraded by lysosomal hydrolases.

Cancer risk factors, including obesity, aging, alcohol abuse, chronic inflammation, and infection, interfere with either the initiation or termination of autophagy to promote cancer development. ULK, unc51-like kinase; PI3KC, class III phosphatidylinositol (PI) 3′ kinase; ATG, autophagy related gene; AMPK, AMP-activated protein kinase; mTORC1, mammalian target of rapamycin complex 1; ER, endoplasmic reticulum; IL-1β, interleukin 1β; ROS, reactive oxygen species; HCV, hepatitis C virus; HBV, hepatitis B virus; EBV, Epstein-Barr virus; KSHV, Kaposi’s sarcoma-associated herpesvirus; cFLIP, viral FLICE inhibitory protein; vBcl-2, viral B cell lymphoma 2; EBNA1, EBV nuclear antigen 1; VacA, vacuolating cytotoxin A; HBx, HBV x protein; H. pylori, Helicobacter pylori. Mechanistic details are provided in the main text.

Homeostasis and rapid adaptation to environmental changes are key to organismal health and survival. Autophagy, a “self-eating” process that clears intracellular waste, attenuates cell stress and keeps pro-carcinogenic processes at bay. These tumor-suppressive functions of autophagy are well recognized and recently reviewed (). While many previous studies had focused on the cell-autonomous nature of autophagy, it has become clear that autophagy-dependent tumor suppression is also executed through downregulation of chronic tumor-promoting inflammation and/or enhancement of anti-tumor immunity. By surveying the relationships between autophagy, inflammation, and immunity (the “AII Troika”), this Review aims to explain how they shape the immune landscape that modulates malignancy. We will discuss how these basic concepts can be translated to improve cancer immunotherapy.

In addition to cell-autonomous modulation of cancer cell physiology, systemic and local autophagy defects, caused by various cancer risk factors, also affect cancer-associated inflammation and immune responses ( Figures 1 and 2 ). As described in detail below, defective autophagy in myeloid cells enhances tumor-promoting inflammation while compromising antigen presentation. Conversely, stimulation of autophagy suppresses tumor-promoting inflammation and enhances anti-cancer immunity ( Figure 2 ). The interaction and crosstalk between members of the “AII Troika,” their effects on cancer development/progression, and the response to immunotherapy are the focal points for this review.

Numerous cancer risk factors (e.g., aging, obesity, and chronic inflammation) interfere with the proper functioning of the autophagic machinery ( Figure 1 ). In epithelial cells, defective autophagy can promote tumor initiation by enhancing oxidative stress and genomic instability, as well as by activating transcription factor NRF2 (nuclear factor erythroid 2-related factor 2), which paradoxically induces expression of genes encoding anti-oxidant and drug-metabolizing enzymes (). Defective autophagy also interferes with oncogene-induced senescence, an important tumor-suppressive mechanism, and thereby leads to uncontrolled proliferation of cancer progenitor cells (). Conversely, once the malignant phenotype has been established, autophagy serves as a survival mechanism that provides rapidly proliferating cancer cells with nutrients (). Nonetheless, cancers in which autophagy is upregulated, as indicated by accumulation of microtubule-associated protein 1A/1B light chain 3 (LC3) puncta, exhibit higher density of CD8T cells and lower number of Foxp3T regulatory cells (Treg) in the tumor bed (). Thus, enhanced autophagy correlates with activation of anti-tumor immunity, and its downregulation may allow malignant growths to avoid immune surveillance. Indeed, oncogene activation can inhibit autophagy, in part through a mechanism similar to one used for inhibition of apoptosis (). Further complicating the intricate power balance within the “AII Troika,” rapid tumor growth results in hypoxia and necrosis at the tumor core, leading to DAMP release and recruitment and activation of macrophages and dendritic cells (). Stimulation of autophagy may suppress this inflammatory response that drives tumor growth by promoting the survival of hypoxic and nutrient-starved cancer cells and by clearing damaged mitochondria ( Figures 2 and 3 ).

Overview of the cancer governing “AII” troika and how its function is modified by certain cancer-associated processes. MHC I, major histocompatibility complex I; ICD, immunogenic cell death; DAMP, damage-associated molecular patterns; NLRP3, nod-like receptor pyrin domain containing protein 3.

The presence of LC3B puncta and HMGB1 expression in malignant cells correlate with the immune infiltrate in breast cancer.

The ultimate cancer risk factor is old age, which has a detrimental effect on autophagy (). Defective autophagy in aged individuals or animals results in aberrant clearance of damaged mitochondria, leading to elevated inflammation and accumulation of ROS and protein aggregates that cause ER stress (). These cellular defects contribute to different degenerative diseases and enhance tumor initiation and malignant progression (). Aging is accompanied by indolent inflammation and parainflammation manifested by increased basal production of IL-1β, IL-18, TNF, and IL-6 (). All of these cytokines enhance cancer development and progression (). Chronic inflammation also compromises anti-cancer immunity ().

Obesity causes nonalcoholic fatty liver disease (NAFLD) that can progress to nonalcoholic steatohepatitis (NASH), which increases HCC risk (). Ongoing autophagy reduces accumulation of lipid droplets in hepatocytes, thus providing a safeguard against NAFLD and NASH (). Conversely, obesity suppresses autophagy by multiple mechanisms, including activation of calcium-dependent protease calpain-2 that leads to ATG7 degradation (), activation of mammalian target of rapamycin complex 1 (mTORC1), and inhibition of unc-51 like autophagy activating kinase 1 (ULK1) activity () or defective autophagosome-lysosome fusion due to changes in membrane lipid composition and ER stress (). Obesity also compromises macrophage autophagy (), which can result in enhanced IL-1β and IL-18 production (). Consistent with this notion, myeloid-specific Atg5 ablation greatly increases the likelihood of NASH development upon consumption of high-fat diet (). Steatotic hepatocytes release linoleic acid that causes depletion of liver resident CD4T cells, thereby contributing to the dismanteling of antitumor immunity ().

Another life-threatening inflammatory disease associated with insufficient autophagy is chronic pancreatitis (). Chronic pancreatitis greatly increases pancreatic cancer risk and so do other pancreatitis and pancreatic cancer risk factors, such as alcohol consumption and high caloric intake, all of which interfere with autophagy activity (). Genetic manipulations that attenuate with autophagy lead to chronic pancreatitis in mice (). Alcohol abuse also causes alcoholic hepatitis (ASH), which is accompanied by lipid droplet accumulation and a marked increase in HCC risk. Ethanol metabolism interferes with AMP-activated protein kinase (AMPK) activity, thereby compromising autophagy (). Moreover, ethanol metabolism elicits mitochondrial damage, causing ROS production and hepatocyte death (). Dying hepatocytes release DAMP that trigger NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation in resident liver macrophages, resulting in neutrophil infiltration and ASH development (). Defective myeloid cell autophagy enhances NLRP3 inflammasome activation, thereby augmenting IL-1β production, neutrophil infiltration, and liver damage ().

The role of AMP-activated protein kinase in the action of ethanol in the liver.

Infectious agents that do not produce oncoproteins stimulate cancer development by inducing chronic tumor-promoting inflammation. Inflammatory bowel diseases (IBD), including ulcerative colitis (UC) and Crohn’s disease (CD), greatly increase the risk of colorectal cancer (CRC) due to elevated expression of inflammatory cytokines, such as IL-6, TNF, and IL-1β (). Autophagy related 16-like 1 (ATG16L1) maps to a CD susceptibility locus that is associated with an elevated CRC risk (). ATG16L1 deficiency afffects the initiation of xenophagy () and enhances expression of Laptm5, a lysosomal transmembrane protein that promotes A20 degradation, thereby potentiating nuclear factor-kappa B (NF-κB) signaling and dendritic cell (DC) activation (). Due to enhanced inflammasome activity, ATG16L1-deficient macrophages produce high amounts of IL-1β and IL-18 (), two cytokines known to increase CRC risk. ATG16L1 deficiency also compromises Paneth cell maturation, resulting in decreased production of antimicrobial peptides (), which, together with defective xenophagy contribute to high microbial load in the lamina propia. The increase of gut microbe translocation can stimulate IL-23 and IL-17 production that promote CRC development and progression ().

Age-related, metabolic, and environmental factors, as well as infectious agents that increase cancer risk, may do so by interfering with the initiation or completion of autophagy ( Figure 1 ). Unlike laboratory-induced ablation of autophagy related genes (ATG) defects, cancer risk factors do not block autophagy completely, but over time, even a partial decrease in autophagic flux will result in chronic pathologies and elevated cancer risk due to accumulation of cellular waste. Cancer-causing infectious agents, including Helicobacter pylori, Epstein-Barr virus (EBV), Hepatitis B virus (HBV), and Kaposi’s sarcoma-associated Herpesvirus (KSHV), have evolved multiple strategies to avoid xenophagic elimination (). These pathogens produce bacterial and viral proteins that bind lysosomes and alter their acidification and ability to degrade autophagosome-delivered cargo. For instance, H. pylori produces vacuolating cytotoxin A (VacA) that blocks autolysosome function to promote its own survival and protect the cytotoxin-associated gene A (CagA) protein from degradation (). Stabilized CagA spreads to other host cells, where it activates signaling pathways that stimulate motility and proliferation and induce gastric mucosal metaplasia (). Although it is not entirely clear how H. pylori promotes gastric cancer development, CagA-induced changes in the behavior of gastric epithelial cells and accumulation of p62/SQSTM1 and reactive oxygen species (ROS) in the gastric mucosa of patients infected with virulent VacA-producing strains are thought to be of importance (). In a similar vein, EBV nuclear antigen 1 (EBNA1) blocks autophagy by inhibiting lysosome acidification, thereby evading xenophagy, a “selective” form of autophagy that eliminates cell-invading microbes. Defective autophagy also impairs EBNA1 presentation by major histocompatibility class II (MHC-II) molecules, an autophagy-dependent process (). Furthermore, dysregulation of autophagy results in cytosolic accumulation of the EBV oncoprotein latent membrane protein 1, LMP1, which activates signaling pathways that control cell proliferation and survival, and inhibits tumor suppressors (). In contrast, HBV, which greatly increases liver cancer risk, produces the small surface protein (HBs) that triggers the unfolded protein response (UPR), resulting in stimulation of autophagy (). Moreover, another HBV viral protein, HBx (HBV X protein), binds to class III PI 3-kinase, VPS34, and causes persistent stimulation of autophagy initiation that promotes HBV core protein maturation (). However, HBx also interacts with V-ATPase and impairs lysosome acidification and proteolysis (). This results in accumulation of p62/SQSTM1 and viral proteins in infected hepatocytes, increasing their likelihood to die (). Enhanced hepatocyte death and subsequent compensatory proliferation strongly enhance hepatocellular carcinogenesis (). By activating NRF2, p62 accumulation protects hepatocellular carcinoma (HCC)-initiating cells from ROS-induced death, allowing them to accumulate numerous oncogenic mutations (). Hepatitis C virus (HCV), on the other hand, induces endoplasmic reticulum (ER) stress to interfere with autophagy (). In addition, HCV viral protein NS4B interacts with VPS34 and Rab5 to inhibit autophagosome maturation and autolysosome formation, although the precise mechanism remains unknown (). Interestingly, certain viruses encode viral proteins that are homologs of important regulators of autophagy. For instance, KSHV encoded-protein vFLIP (viral FLICE inhibitory protein) and vBcl-2 (viral B cell lymphoma 2) that interact with Atg3 and Beclin1, respectively, block autophagosome formation or vesicle nucleation ().

Another mechanism through which autophagy squelches the tumor-promoting inflammatory fire is xenophagy. For instance, following infection of phagocytes with H. pylori, Salmonella, Listeria, or Shigella, PAMP recognition by TLR and NLR stimulates xenophagy, which, by reducing pathogen load, attenuates microbe-induced inflammation (). Like damaged mitochondria, intracellular bacteria are recognized by autophagy receptors such as p62, NBR1, and NDP52, which promote their autophagic clearance (). One trigger of xenophagy could be energy imbalance, caused by competition between the invading microorganism and the host cell for nutrients, which results in AMPK activation and inhibition of mTORC1, stimulating the initiation of autophagy through modulation of ULK1/2 phosphorylation (). Intriguingly, xenophagy and mitophagy are evolutionarily related, as mitochondria are thought to have originated from endosymbiotic bacteria (). Thus, the control of inflammation, by either exogenous or endogenous insults, may be an ancient function of autophagy in multicellular organisms. Given the importance of inflammation in tumor development (), there is little doubt that the anti-inflammatory function of autophagy makes a key contribution to its tumor suppressive ability.

Given its dependence on mitochondrial damage, NLRP3 inflammasome activation is negatively regulated by autophagy, which keeps IL-1β and IL-18 production and subsequent inflammation in check (). Mitochondria that have been damaged upon macrophage encounter with NLRP3 activating stimuli undergo Parkin-dependent ubiquitylation, followed by recruitment of the ubiquitin-binding autophagy adaptor p62/SQSTM1 ( Figure 3 ). By recognizing LC3 on phagophore membranes, p62 ensures the mitophagic elimination of damaged mitochondria and termination of NLRP3 inflammasome activation (). Of note, p62 gene transcription is induced by NF-κB, the same process that controls the first step in IL-1β production and accounts for upregulation of NLRP3 itself. Genetic ablation of p62 or inhibition of IκB kinase β (IKKβ), which attenuates p62 induction, results in excessive secretion of IL-1β and IL-18 by macrophages that have been presented with NLRP3 activating stimuli, despite the concomitant decrease in Il1b gene transcription (). Enhanced IL-1β secretion results in excessive inflammation and neutrophilia (). Of note, the NLRP3 inflammasome likely plays an important and rather general role in the onset of tumor promoting inflammation, as it is activated by carcinogenic asbestos and silica microparticles (), as well as by lipids and cholesterol, which stimulate NAFLD progression to NASH and thereby increase HCC risk.

The ability of autophagy to prevent excessive inflammation was first observed in mice rendered deficient in Atg16l1, which produce significantly more IL-1β than wild-type (WT) controls and are more vulnerable to septic shock (). Further studies demonstrated that anything that blocks autophagy, be it Atg gene ablation or pharmacological intervention, results in enhanced caspase-1 activation and increased IL-1β production and secretion (). Caspase-1 is activated after its incorporation into large protein assemblies called inflammasomes, which also contain a sensor protein that belongs to the NLR (Nod-like receptor) family and the adaptor protein ASC (apoptosis-associated speck-like). Production of biologically active IL-1β, one of the two most potent inflammatory cytokines (the other being TNF), depends on two steps. First, IL-1β gene transcription is induced upon NF-κB activation caused by PAMP or DAMP binding to Toll-like receptors (TLR) or engagement of TNF receptors. Translation of IL-1β mRNA results in production of precursor pro-IL-1β, which cannot be secreted by macrophages. The second step is inflammasome dependent and is initiated by NLR-specific stimuli (). For instance, NLRC4 (NLR family CARD domain-containing protein 4) senses cytosolic flagellin, whereas NLRP1b (NLR Family, Pyrin Domain Containing 1) detects anthrax lethal toxin. The sensor protein with the broadest sensitivity is NLRP3, which responds to a panoply of stimuli, including ATP, toxins (e.g., nigericin), uric acid, cholesterol, alum, silica and asbestos microcrystals, and lipid particles. Notably, none of these stimuli is directly recognized by NLRP3. Instead they either open potassium channel(s), disrupt membrane integrity, or function through other indirect mechanisms that somehow culminate in mitochondria damage (). Damaged mitochondria release or present substances such as mtROS, oxidized mitochondrial DNA (mtDNA), or cardiolipin, which are presumed to be direct NLRP3 inflammasome activators (). Although the identity of the ultimate NLRP3 ligand and how it activates the inflammasome remain to be determined, it is quite well established that macrophages encountering NLRP3 activating stimuli display mitochondrial damage and that elimination of mitochondria or mtROS specifically prevents NLRP3 inflammasome activation and production of mature IL-1β and IL-18 () ( Figure 3 ).

Stimulation of macrophages with DAMP (e.g., ATP and uric acid), carcinogenic particles (asbestos and silica microfibers/crystals), or PAMP (e.g., bacterial toxins) results in mitochondrial damage that is characterized by loss of mitochondrial membrane potential and subsequent release of mtDNA and mtROS. These mitochondrial signals in turn activate the NLRP3 inflammasome to induce IL-1β and IL-18 secretion. Loss of mitochondrial membrane potential (ψm) activates PINK1, a mitochondrial protein kinase that phosphorylates ubiquitin chains attached to mitochondrial outer membrane proteins. Phosphorylated ubiquitin interacts with and activates Parkin, an E3 ubiquitin ligase that further ubiquitinates mitochondrial outer membrane proteins. Ubiquitin-tagged mitochondria are recognized by the UBA domain of p62, whose expression is induced upon NF-κB activation. p62 also binds to LC3 and targets ubiquitinated mitochondria to autophagosomal clearance. By eliminating signal-emitting mitochondria, macrophage limits the extent of NLRP3 inflammasome activation. MSU, monosodium urate crystal; PINK1, PTEN-induced putative kinase 1; LC3, microtubule-associated protein 1A/1B light chain 3; NF-κB, nuclear factor-kappa B; UBA, ubiquitin association domain.

In addition to xenophagy that reduces tumor-promoting inflammation by eliminating pathogen-associated molecular patterns (PAMP)-producing intracellular microbes, other forms of autophagy also suppress tumor-promoting inflammation. In addition to increasing cancer risk, sustained and/or unresolved inflammation causes collateral tissue damage, doing more harm than good. Conversely, a properly mounted, focused, and transient inflammation promotes tissue repair and regeneration (). As discussed below, autophagy ensures a well-balanced inflammatory response that is accompanied by restoration of homeostasis.

Autophagy also stimulates thymic “negative selection” of autoreactive CD4T cells, thereby maintaining central T cell tolerance (). Autophagy further regulates lymphocyte development and functional diversification. Naive T cell number is dramatically reduced in the absence of mitophagy, and mature T cells require autophagy for survival (). Additionally, autophagy indirectly influences Th17 cell polarization by restraining IL-1β production by innate immune cells. Since IL-1β promotes Th17 lineage commitment together with IL-6 and TGFβ (), defective autophagy should enhance IL-17 production and tumorigenesis (). As mentioned above, defective hepatocyte autophagy results in accumulation of lipid droplets, which may promote the release of linoleic acid, causing the depletion of hepatic CD4T cells and creating an immunosuppressive microenvironment that promotes cancer growth (). The generation of T-cell-dependent and -independent antibody responses also requires functional autophagy as its absence results in ER stress and consequent plasma cell death (). In summary, autophagy tunes down inflammation while boosting adaptive immunity capable of curtailing tumor growth and progression.

Adaptive immunity relies on recognition of extracellular (exogenous) or intracellular (endogenous) peptide epitopes presented by MHC class II and I molecules that are recognized by CD4and CD8T cells, respectively (). Autophagy in antigen-presenting cells (APC) can promote antigen presentation by both MHC class II and I molecules. For instance, upon uptake of extracellular antigens (e.g., microbial or tumor antigens) by APC, autophagy promotes trafficking of the engulfed antigens to endosomes, where they are digested by cathepsins and loaded onto MHC class II molecules that eventually translocate to the plasma membrane and present antigens to CD4T cells. Although the precise mechanism remains to be further investigated, autophagy also facilitates “cross-presentation” of exogenous constituents by facilitating their loading onto MHC class I molecules that ultimately activate antigen-specific CD8T cells. Notably, autophagy-mediated cross-presentation is important for mounting T cell responses under various stressed conditions, when proteasome function is compromised and antigenic peptides cannot be imported to the ER for loading onto MHC class I molecules. In support of this notion, in tumor-bearing mice or cancer patients, tumor-infiltrating APC found in the draining lymph nodes are often functionally compromised. To ensure the generation of an effective antitumor cytotoxic T cells (CTL) response, APC autophagy then becomes of utmost importance and needs to be stimulated to facilitate processing and cross-presentation of tumor antigenic peptides by MHC class I molecules (). Furthermore, autophagy also induces upregulation of MHC class I molecules in response to IFN-γ (), further enhancing the “cross-presentation” of extracellular antigens (). Last but not least, in addition to its roles in APC, autophagy in cancer cells can indirectly promote “cross-presentation” of tumor antigens by facilitating their release from dying cells, thereby increasing extracellular antigen availability ().

Autophagy and Its Split Personality: Modulation of Immunotherapy

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et al. Activation of the NLRP3 inflammasome in dendritic cells induces IL-1beta-dependent adaptive immunity against tumors. ICD also results in ATP release, a process considered to depend on activation of autophagy. Extracellular ATP functions as a “find-me” signal that, by engaging the purinergic receptor P2Y2, recruits monocytes that, upon maturation into macrophages/DCs, engulf dying cancer cells. By binding a related purinergic receptor, ligand-gated ion channel 7 (P2X7), extracellular ATP activates the NLRP3 inflammasome to induce IL-1β secretion, a process critical for a successful CTL response (). Of note, the preferential engagement of distinct purinergic receptors depends on ATP concentration: at <1 μM, ATP predominantly binds P2Y2 to induce monocyte infiltration, whereas at >100 μM, it engages P2X7 to activate the NLRP3 inflammasome (). Last but not least, HMGB1 is also required for ICD induction by chemotherapeutic agents and subsequent activation of anti-tumor immunity as shown in colorectal cancer (). HMGB1 released from cancer cells undergoing ICD is thought to engage TLR4 on APC, resulting in enhanced CD8T cell infiltration, which, in combination with immune checkpoint inhibitors, leads to a strong antitumor response (). In addition, HMGB1-TLR4 signaling can prime the NLRP3 inflammasome, which further enhances induction of an antitumor CTL response ().