Autophagy proteins influence endocytosis for MHC restricted antigen presentation

Accepted Manuscript Title: Autophagy proteins influence endocytosis for MHC restricted antigen presentation Author: Christian Munz ¨ PII: DOI: Reference:

S1044-579X(19)30021-5 https://doi.org/10.1016/j.semcancer.2019.03.005 YSCBI 1551

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Seminars in Cancer Biology

Received date: Revised date: Accepted date:

21 January 2019 19 March 2019 25 March 2019

Please cite this article as: Munz ¨ C, Autophagy proteins influence endocytosis for MHC restricted antigen presentation, Seminars in Cancer Biology (2019), https://doi.org/10.1016/j.semcancer.2019.03.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Christian Münz

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Autophagy proteins influence endocytosis for MHC restricted antigen presentation

Christian Münz* Viral Immunobiology, Institute of Experimental Immunology, University of Zurich, Switzerland

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*Address correspondence to: Christian Münz, Viral Immunobiology, Institute of Experimental Immunology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland, e-mail: [email protected]

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Abstract

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T cells of the adaptive immune system monitor protein degradation products via their presentation

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on major histocompatibility complex (MHC) molecules to recognize infected cells. Both macroautophagy and endocytosis target intra- and extracellular constituents, respectively, for

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lysosomal degradation. This results in antigen processing for MHC presentation and influences the trafficking of MHC molecules. This review will discuss recent evidence that the molecular

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machinery of macroautophagy regulates also endocytosis at the level of phagosome maturation and cell membrane internalization. These non-canonical functions of this machinery affect both

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MHC class I and II restricted antigen presentation to CD8+ and CD4+ T cells, respectively, and

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should be harnessed to improve immune responses against infectious diseases and cancer.

Keywords: LC3 associated phagocytosis (LAP), MHC class I internalization, MHC class II antigen

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processing, CD8+ T cell responses, CD4+ T cell stimulation

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1. Introduction

T cells are the cornerstone of adaptive immune responses. They coordinate both cellular and humoral immunity by providing T cell help for both antibody producing B cells and cytotoxic lymphocytes (1). For their stimulation to fulfill these functions T cells need to detect antigen derived peptides on major histocompatibility complex (MHC) molecules (2, 3). These are octa- or nonameric peptides presented on MHC class I molecules for cytotoxic CD8+ T cells, and longer

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peptides with a nonameric core sequence for presentation on MHC class II to helper CD4+ T cells. The respective peptides are primarily generated by proteasomal proteolysis in the cytosol and nucleus for MHC class I presentation and by lysosomal hydrolysis for MHC class II presentation. MHC class I ligands are then transported into the endoplasmic reticulum (ER) via the transporter associated with antigen processing (TAP) and are loaded in a chaperone and disulfide isomerase

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dependent fashion onto MHC class I molecules in the MHC class I loading complex (2). Stable MHC class I complexes travel then to the cell surface for surveillance by CD8+ T cells. In contrast, MHC class II molecules are prevented from peptide loading in the ER by the invariant chain (Ii), which blocks the peptide binding groove and directs MHC class II molecules to late endosomes, called the MHC class II containing compartment (MIIC) (4). There, both Ii and the antigen is degraded by lysosomal proteolysis, allowing for high affinity peptide loading with the assistance of the chaperone HLA-DM (H2-M in mice). Stable MHC class II peptide complexes then are

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transported to the cell surface for stimulation of CD4+ T cells. Thus, access of antigens to

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proteasomal or lysosomal degradation and transport of the resulting peptides to MHC molecules or vice versa MHC molecules to the source of their ligands determines efficient antigen processing

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for MHC presentation. Classically, antigen access to proteasomal degradation requires antigen

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expression in the presenting cell after for example viral infection. However, in specialized antigen presenting cells, like dendritic cells, endocytosed extracellular material can also gain access to the cytosol in an antigen processing pathway leading to cross-presentation on MHC class I

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molecules (2). Vice versa, lysosomal degradation is usually accessed by endocytosed

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extracellular antigens, but autophagy, a set of catabolic pathways that deliver cytoplasmic material for lysosomal degradation, can promote intracellular antigen presentation on MHC class

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II molecules (5-7) (Figure 1).

At least three autophagy pathways exist in eukaryotic cells. These are chaperone-

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mediated autophagy (CMA), microautophagy and macroautophagy (8). During CMA, chaperone binding substrates get transported across lysosomal membranes for degradation (9). This requires cytosolic and luminal HSC70 members, LAMP2a in the membrane and a KFERQ like

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sequence in the substrate for HSC70 interaction. Microautophagy also delivers HSC70 binding cargo with a similar recognition sequence across lysosomal and late endosomal membranes. However, during microautophagy these membranes invaginate and form intraluminal vesicles that are then degraded. Finally, for macroautophagy more than 40 autophagy-related gene (atg) products are required that build a double membrane vesicle, the autophagosome, engulfing large cytoplasmic structures, including organelles like mitochondria, and then fuse with late endosomes or lysosomes for cargo and inner membrane degradation (10, 11). For this purpose, the more

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than 40 Atg proteins assemble into complexes which modify membranes for autophagic membrane formation, substrate recruitment into these membranes and their fusion with late endosomes or lysosomes. A first complex that incorporates metabolic signals to stimulate macroautophagy contains the unc51-like autophagy-activating kinase 1 (ULK1) at its center. In addition, Atg13, FIP200 and Atg101 belong to this complex. ULK1 complex activity is stimulated

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by phosphorylation by the AMP-activated protein kinase (AMPK), which senses energy loss via AMP (Figure 1). Vice versa the ULK1 complex is inhibited by phosphorylation via the target of rapamycin complex (mTORC1), which is stimulated upon growth signaling and energy surplus. The ULK1 complex in turn activates via phosphorylation the class III phosphoinositide complex (PI3K) containing VPS34, beclin 1, Atg14L and the beclin 1 regulator AMBRA1. This PI3K complex sets the first membrane mark at the location of autophagosome generation. This PI3 mark then recruits via the WD repeat domain phosphoinositide-interacting protein 2 (WIP2) the

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autophagy protein Atg16L1 at sites at which also the omegasome protein zinc-finger FYVE

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domain-containing protein 1 (DFCP1) can be found. Atg16L1 forms a complex with the Atg5Atg12 conjugate that is assembled in a ubiquitin-like reaction with the help of Atg7 and Atg10.

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The Atg16L1/Atg5-Atg12 complex has E3-like activity for other ubiquitin-like proteins, the Atg8

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family, whose most prominent member is LC3B and contains also GABARAP proteins (Figure 1). Atg8 proteins get initially processed at their C-terminus by the protease Atg4, liberating a glycine residue, that then in a ubiquitin-like reaction with Atg7 as E1-like and Atg3 as E2-like enzymes

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gets coupled to phosphatidylethanolamine (PE) in the forming inner and outer autophagosome

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membrane. The LC3B-PE conjugate migrates faster in SDS-polyacrylamide gel electrophoresis (LC3-II) and can be used to quantify autophagic membranes. These Atg8 proteins mediate the

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elongation of the autophagosomal membrane, possibly with Atg9 containing vesicles, and form membrane anchors for autophagic substrate recruitment. This substrate recruitment occurs via LC3-interacting regions in proteins, some of which bind ubiquitinated cargo, like p62

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(sequestosome 1) (Figure 1), optineurin, NDP52, TAXBP1 and NBR1. The Atg8s are also involved in the autophagosome closure, upon which the Atg8s are recycled from the outer autophagosome membrane by proteolytic cleavage via Atg4. These completed autophagosomes

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are then ready to fuse with late endosomes and lysosomes in a syntaxin 17, YKT6 and Rab7 dependent fashion (12, 13). Direct binding of ULK1 and the inhibitor of apoptosis protein BRUCE to syntaxin 17 have recently been found to respectively inhibit or activate autophagosome fusion with lysosomes (14, 15). The resulting autolysosome then degrades both cargo and inner autophagosomal membrane. Therefore, LC3-II and p62 turnover can be used to assess autophagic flux. Targeting viral and tumor antigens into this autophagic flux via fusion to the N-

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terminus of LC3B enhances MHC class II presentation by up to 20 fold (16-19). However, the membrane modifications that are performed by the class III PI3K complex and the Atg16L1/Atg5Atg12 complex are not only used for autophagosome generation. In recent years it has also been appreciated that they regulate endocytic processes. This review will discuss this recent literature on the non-canonical role of Atg proteins during phagosome maturation and internalization from

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the cell membrane.

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2. Regulation of phagocytosis by autophagy proteins The non-canonical regulation of phagocytosis by Atg proteins has been coined LC3-associated phagocytosis (LAP) (20). It was originally observed during the uptake of yeast cell wall components by macrophages. Engagement of the pathogen associated molecular pattern receptors toll-like receptors (TLRs), in particular TLR2, but also to some extent TLR4, during the

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phagocytosis of these yeast components resulted in single membrane surrounded phagosomes that were decorated with LC3-II at their cytosolic side (20-22) (Figure 1). TLR stimulation does not only stabilize components of the macrautophagy machinery at phagosomes, but also upregulates their expression as well as lysosomal function. Along these lines it has been demonstrated that TLR engagement leads to nuclear localization of the basic helix-loop-helix transcription factor 3 (TFE3) that together with transcription factor EB (TFEB) from the same protein family induces expression of macroautophagy and lysosomal proteins (23). Additional

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receptors that can stimulate LAP include antibody Fc receptors, the C-type lectin Dectin-1 and

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the phosphatidylserine binding receptor TIM4, that engages apoptotic cellular debris (24, 25). LAP does not require the ULK1 complex, but modifies its phagosomal membrane also by PI3

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phosphorylation with the class III PI3K complex, containing VPS34 and beclin 1 (20, 26) (Figure

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1). In contrast to autophagosome formation, the LAP associated PI3K complex does, however, not contain Atg14 and AMBRA, but requires the UV radiation resistance associated (UVRAG) and the Rubicon protein (26), a PI3K complex that was previously described to inhibit autophagosome

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fusion with lysosomes (27-29). This could suggest that the PI3K complex containing UVRAG is

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required for LAP as well as autophagosome fusion with lysosomes, and the one containing Atg14 for autophagosome formation. The resulting PI3P modification of the phagosomal membrane

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facilitates the recruitment of the NADPH oxidase 2 (NOX2), which is required for LAP (21, 26, 30) (Figure 1). Indeed, macrophages from patients with chronic granulomatous disease that carry loss-of-function mutations in NOX2 are not able to perform LAP, but can assemble

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autophagosomes (21). While NOX2 is required for LAP, it is not specific for this process. Most phagosomes recruit NOX2 and their pH maturation is attenuated via reactive oxygen species (ROS) production by this enzyme (31), but only a subset (< 10%) will recruit LC3B to its membrane

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(21). This subset of phagosomes conjugates LC3B after recruitment of the Atg16L1/Atg5-Atg12 complex, but this recruitment seems to be PI3P independent and instead mediated by the lipid binding WD40 domain of Atg16L1 (32) (Figure 1). Therefore, the function of the required ROS production by NOX2 during LAP remains unclear. Conjugation of LC3B to phagosomes influences their maturation and trafficking, possibly via the interaction with components of the transport machinery along microtubules (33-35). In mouse macrophages LAP was found to accelerate

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fusion with lysosomes (20) and mice deficient in LAP components accumulate apoptotic vesicles, leading to lupus erythematosus-like systemic autoimmunity (36). In contrast, in human macrophages and dendritic cells LAP seems to delay phagosome fusion sometimes for hours to maintain endocytosed antigen (21). Finally, in plasmacytoid dendritic cells, the main type I interferon producers during many viral infections, and in B cells LAP might reroute phagocytosed

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material to TLR containing endosomes for more efficient immune detection of pathogens (37). (38). Alternatively, the B cell receptor requires the macroautophagy machinery for internalization into late endosomes, possibly with secretory lysosome characteristics for its polarization towards immobilized antigen (39, 40). Interaction of Atg16L1 and GABARAPs with the centrosomes via the pericentriolar material 1 (PCM1) protein could be involved in B cell receptor containing secretory lysosome polarization (40, 41) and Atgs have indeed been suggested to facilitate secretory lysosome release in osteoclasts (42). These functions might be important during

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germinal center reactions for efficient antibody responses during viral infections and autoimmunity

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(43, 44). Thus, Atg proteins modulate phagosome fate in a cell type specific manner. Only a subset of the molecular machinery of macroautophagy is required for LAP, including the PI3K and

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the Atg16L1/Atg5-Atg12 complexes, but without the help of the ULK1 complex. However, in

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addition to these Atg proteins NOX2 and possibly a different composition of the PI3K complex, including Rubicon, is required for LAP. Since this PI3K complex including UVRAG seems to regulate both extracellular and intracellular antigen processing via LAP and autophagosome

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maturation, respectively, it might be essential for MHC class II restricted antigen presentation

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during cancer immunotherapy.

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3. LC3 associated phagocytosis during antigen processing for MHC class II presentation In addition to degrading more efficiently pathogens and apoptotic bodies, or rerouting phagocytosed cargo to TLRs for more efficient immune stimulation (20, 36-38), LAP has also been shown to contribute to MHC class II presentation of phagocytosed antigens (21, 25, 32) (Figure 1). Yeast antigen formulations were initially used to show this LAP contribution to antigen

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processing for MHC class II presentation (21, 25). These were Candida albicans antigens and the model antigen ovalbumin expressed by Saccharomyces cerevisiae. Their presentation to antigen specific CD4+ T cells, initially producing IL-17, but with extended clonal in vitro culture also IFN- and GM-CSF, was compromised after phagocytosis, if the machinery of LC3 lipidation was down-modulated (21, 25). In the case of human macrophages and dendritic cells especially the prolonged MHC class II presentation of yeast derived antigens was compromised in the absence of LAP components (21). This lack of extracellular antigen presentation on MHC class II

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molecules has now also been extended to a model antigen consisting of green fluorescent protein

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(GFP) fused to the alpha chain of the mouse H2-E molecule. Presentation of the H2-E MHC class II derived peptide on the H2-Ab MHC class II molecule can be evaluated with a specific antibody

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(YAe), and it was found that mouse dendritic cells present H2-E derived peptides less well on

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MHC class II when they lack the WD40 domain of Atg16L1 that is required for LAP (32). Furthermore, also in vivo ovalbumin coated on injected splenocytes gets less well presented to CD4+ T cells in the absence of Atg5 in dendritic cells (22). In a second in vivo study Atg5 deficiency

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in dendritic cells also prevented CNS autoimmunity after adoptive transfer of activated CD4+ T

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cells specific for the brain autoantigen myelin oligodendrocyte glycoprotein (MOG) (45). Since MOG is not expressed in dendritic cells its processing for MHC class II presentation after

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phagocytosis was suggested to be mediated by LAP. Indeed, dendritic cells that had engulfed apoptotic oligodendrocytes stimulated MOG specific CD4+ T cells only efficiently when they could perform LAP and expressed Atg5. Consistent with these findings bacterial outer membrane

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vesicles (OMVs) do not elicit IL-10 production in co-cultures of Atg16L1 deficient dendritic cells with CD4+ T cells, nor stimulate immunosuppressive activity by regulatory CD4+ T (Treg) cells

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(46). This extends also to the destabilized atg16L1T300A mutant that is associated with inflammatory bowel disease (IBD). While Atg16L1, Atg5, Atg7 and Rubicon in dendritic cells are required for CD4+ Treg stimulation, ULK1, FIP200 and Atg14 are not. This points towards a role of LAP in OMV derived antigen presentation on MHC class II molecules towards CD4+ Treg cells. Along these lines TLR2 was also required for this OMV induced CD4+ Treg stimulation, as was NOD2 which has been shown to be required for Atg protein dependent CD4+ T cell stimulation by dendritic cells (47). However, NOD2, in contrast to TLR2, has not yet been implicated in LAP

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directly. Moreover, OMV injections did not protect from colitis in the absence of Atg16L1 in dendritic cells, and also IL-10 production was significantly impaired in vivo. Furthermore, macrophage dependent immune suppression required also LAP rather than macroautophagy in the tumor microenvironment and in a T cell dependent fashion (48). Thus, LAP seems to support yeast, bacterial, auto- and model antigen processing for MHC class II restricted antigen

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presentation to effector and regulatory CD4+ T cells. However, the polarization of the resulting CD4+ T cell responses might rather be determined by the cytokine milieu resulting from differential

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antigen presenting cell activation than the pathway of antigen processing.

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4. Regulation of receptor endocytosis by autophagy proteins Besides this role of LC3 conjugation at the cytosolic side of phagosomes in controlling their trafficking fate, components of the macroautophagy machinery have also been implicated in the internalization of surface molecules and viruses (49, 50). This was first reported for the amyloid precursor protein (APP), whose proteolytic product, the A peptide, accumulates in protein

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plaques in brains of Alzheimer disease patients (51). Both starvation and mTORC1 inhibition, which both increase the activity of the ULK1 complex and thereby macroautophagy, increased APP internalization and degradation without A production (52). This degradation was dependent on Atg5 and beclin 1 (52, 53). The APP internalization requires the interaction of lipidated LC3-II with the adaptor protein complex 2 (AP2) that supports clathrin mediated endocytosis (54) (Figure 2). One component of this complex (AP2A1) contains a LIR motif for LC3 binding. The lipidation of LC3 that assists in the recruitment of AP2 to APP for internalization might be dependent on

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direct beclin 1 binding to APP (53). Indeed, it was found that beclin 1 binds to APP via the

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precursor protein’s evolutionary conserved domain (ECD) (53) (Figure 2). This brings the UVRAG, VPS34 and beclin 1 containing PI3 kinase complex in close proximity to APP, which facilitates

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endosome maturation. Moreover, lipidated LC3 might not only recruit AP2 to APP at the cell

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membrane, but clathrin itself has also been described to contain a LIR motif (55). Therefore, APP binding to beclin 1 might initiate the phosphoinositide phosphorylation that support LC3 lipidation

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in proximity of this surface protein. The membrane coupled LC3 then in turn recruits members of the clathrin mediated internalization machinery for efficient APP uptake and degradation.

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This or related internalization pathways have also been exploited by viruses. Clathrin mediated endocytosis of the enteric cytopathic human orphan (ECHO) virus 7 of the

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picornavirus family required components of the PI3K and the LC3 lipidation complexes within the macroautophagy machinery (56). Beclin 1, Atg12, Atg14, Atg16L1 and LC3 were required for ECHO virus 7 entry into intestinal epithelial cells. These Atg proteins were not involved in virus

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attachment. Furthermore, white spot syndrome virus (WSSV) of the Nimavirus family enters crayfish cells by clathrin mediated endocytosis that is GABARAP dependent (57) (Figure 2). This

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entry requires the Atg8 family member GABARAP. GABARAP might facilitate interactions with the cytoskeleton during WSSV entry. In addition, to clathrin mediated endocytosis, WSSV can also enter crayfish cells via macropinocytosis and macropinosomes have previously been reported to be also able to recruit LC3 (58). Indeed, during entry of the human filovirus Ebola virus into human cervical epithelial cells by macropinocytosis LC3 was found to be recruited to the entry sites (59) (Figure 2). RNA silencing of beclin 1, Atg7 or LC3B severely compromised entry and infection efficiency. While LC3B without lipidation was still recruited to cell membrane sites of the

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Rab effector Ankfy1 that is required for macropinocytosis, Ebola virus entry required LC3 conjugation to PE. Ankfy1 was also found to directly bind LC3B (59) (Figure 2). Thus, entry of some viruses by both clathrin mediated endocytosis or macropinocytosis seems to benefit from the PI3K and LC3 lipidation complexes of the macroautophagy machinery, and possibly recruits

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endocytosis factors to the entry site via binding to lipidated Atg8 family members.

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5. Atg regulated endocytosis during antigen processing for MHC class I presentation This regulation of internalization from the cell membrane affects also antigen presentation on MHC molecules. While MHC class II surface levels are unaltered on dendritic cells with deficiencies in components of the LC3 lipidation complex, namely Atg5 and Atg7 (22, 45, 60), classical and non-classical MHC class I expression is increased (60, 61). This increased surface

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expression does not result from elevated transcription or transport to the surface, but instead internalization of the classical MHC class Ia molecules H2-Kb and -Db and the non-classical MHC class Ib molecule CD1d is attenuated in the absence of Atg5 or Atg7 in dendritic cells and some macrophages, including alveolar macrophages. While many other surface molecules, including the co-stimulatory molecules CD86 and CD40, do not seem to be affected by this internalization deficiency, CD80 might also be stabilized on dendritic cell populations (60, 62). In Atg5 or Atg7 deficient dendritic cells it was found that adaptor associated kinase 1 (AAK1), which

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phosphorylates the µ subunit of the clathrin associated AP2 complex (AP2M1) for increased

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activity (63, 64), is not efficiently recruited to MHC class I molecules (60) (Figure 2). AAK1 also binds directly to LC3 and is presumably recruited to MHC class I molecules via lipidated LC3 in

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the cell membrane (60) (Figure 2). Interestingly, AAK1 was also recently described to stimulate

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macroautophagy (65). Confirming its role in MHC class I internalization RNA silencing of AAK1 stabilized MHC class I molecules on the surface of dendritic cells (60). Dendritic cells with such increased MHC class I surface levels stimulated influenza A virus specific CD8+ T cell lines more

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efficiently after infection. Moreover, both influenza A and lymphocytic choriomeningitis virus

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infection of mice with Atg5 deficiently in dendritic cells led to increased CD8 + T cell priming and expansion (60). This also resulted in decreased influenza A virus infection, and the CD8+ T cell

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expansion in the absence of Atg5 or Atg7 in dendritic cells inversely correlated with influenza A virus loads. Consistent with these findings, when Atg5 and Atg7 deficiency was targeted to all macrophage populations, but not dendritic cells, influenza A virus infection was also better

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controlled at late time points (> day 10 p.i.), coinciding with the priming of T cell mediated immune control (66). In contrast, mice with FIP200 or Atg14 deficiency in macrophages showed already resistance to influenza A virus early after infection during the innate phase of the immune

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response, associated with pronounced inflammasome hyperactivation. Such hyperactivation, as seen for CD8+ T cell responses associated with decreased classical MHC class Ia internalization in the absence of Atg5 or Atg7, is also seen for NKT cells in mice with LC3 lipidation deficient dendritic cells (61). Invariant NKT cells recognize glycolipids presented on CD1d, one type of nonclassical MHC class Ib molecules (67). CD1d is also stabilized on the surface of Atg5 deficient dendritic cells and presents then glycolipids more efficiently to NKT cells in vitro. Although CD1d

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ligands get loaded in MIICs and thus their transport to these compartments could benefit from macroautophagy or LAP, the dominant phenotype of Atg deficiency seems to be CD1d surface stabilization and NKT cell stimulation is rather augmented than lost by lack of Atg5. Accordingly, glycolipid injection into mice with Atg5 deficient dendritic cells leads to increased NKT cell derived cytokine production. Moreover, immune restriction of the NKT cell sensitive pathogen

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Sphingomonas paucimobilis is attenuated in mice with CD11c driven Atg5 deficiency (61). This improved immune control was associated with higher NKT cell derived cytokine production in vivo. These studies suggest that both CD8+ T cells and NKT cells are more efficiently activated in the absence of Atg5 and Atg7 dependent classical and non-classical MHC class I internalization.

In contrast to enhanced presentation of intracellular antigens on MHC class I molecules in the absence of LC3 lipidation, cross-presentation of extracellular antigens on MHC class I molecules seems to suffer in the absence of Atg7 and VPS34 (68, 69). This was, however, mainly

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observed for soluble ovalbumin in the case of Atg7 and for cell associated ovalbumin in the case

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of VPS34 deficient dendritic cells. Consistent with this deficiency in cross-presentation, mice with VPS34 deficient dendritic cells controlled adoptively transferred B16 melanoma cells less well

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than those with VPS34 (69). For some of these MHC class I cross-presentation pathways the pool

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of MHC class I molecules in early endosomes that results from surface internalization (70) might be more important than for others. Furthermore, lack of macroautophagy in the antigen donor cell compromises also cross-presentation of viral and tumor antigens (71, 72). This results from the

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release of extracellular vesicles, which can be increased upon inhibition of intracellular antigen

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degradation by proteasomes and simultaneous inhibition of autophagosome degradation by lysosomal inhibitors (73-75). Therefore, decreased MHC class I internalization in the absence of

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the LC3 lipidation machinery might increase intracellular antigen presentation to CD8+ T cells, but

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compromise extracellular antigen cross-presentation.

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6. Conclusions The molecular machinery of macroautophagy enables cells to assemble and bend doublemembranes around cytoplasmic cargo for its degradation in lysosomes. It achieves this membrane remodeling by two successive membrane modifications (PI3 phosphorylation and PE conjugation to Atg8 family members) which in turn recruit effector molecules for autophagosome

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formation and substrate recruitment. As discussed above these membrane modifications are however also able to influence endocytic pathways, mainly phagosome maturation and cell membrane internalization. The compartimentalization of the respective membrane modification complexes into different cellular localizations and functions needs to be better understood. Earlier studies by Yoshinori Ohsumi, who received the Nobel prize in 2016 for his discovery of the molecular machinery of macroautophagy, point towards Atg4 removal from membranes as the rate limiting step for the membrane conjugation of the Atg8 family members (76). How this is

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regulated during endocytosis versus macroautophagy requires further studies. A better

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understanding might, however, enable us to therapeutically target the Atg machinery selectively during macroautophagy or endocytosis. Along these lines the dependency of LAP on NOX2

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dependent ROS production could be pharmacologically targeted to compromise the

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immunosuppressive tumor microenvironment (48).

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Conflict of interest statement The author declares no conflict of interest with the discussed topics. Acknowledgements Research in my laboratory is supported by by Cancer Research Switzerland (KFS-4091-02-2017),

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KFSPPrecision-MS of the University of Zurich, the Vontobel Foundation, the Baugarten Foundation, the Sobek Foundation, the Swiss Vaccine Research Institute, the Swiss MS Society and the Swiss

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National Science Foundation (310030B_182827 and CRSII5_180323).

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Figure legends Figure 1. Macroautophagy and LC3 associated phagocytosis (LAP) contribute to MHC class II restricted antigen presentation. Macroautophagy (on the left) is stimulated by ULK1, which is activated by AMPK and inhibited by mTORC1. ULK1 activates the VPS34/beclin 1 complex that phosphorylates PI3 at sites of autophagosome formation, often the endoplasmic

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reticulum (ER). Via this PI3P mark the Atg5/12/16 complex is recruited to conjugate the Atg8 family members, including LC3, to autophagic membranes for membrane elongation and substrate recruitment, via for example p62. The completed autophagosome recycles LC3 from the outer membrane prior to fusion with late endosomes, in which products of lysosomal degradation are loaded onto MHC class II molecules. Phagocytosed cargo reaches these MHC containing compartments (MIIC) preferentially via LC3 associated phagocytosis (LAP, on the right). Engagement of the surface receptors TLR2, FcR, Dectin1 and TIM4 recruit the

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VPS34/beclin 1 complex for PI3 phosphorylation during LAP. PI3P then recruits NOX2 whose

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reactive oxygen species production is required for LC3 attachment to the cytosolic side of the

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respective phagosomes. LC3 is also recycled from phagosomes prior to fusion with MIICs.

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Figure 2. Surface molecules and viruses use Atg8 family members for their internalization from the cell membrane. From left to right: Firstly, MHC class I molecules recruit AAK1 via lipidated LC3 of the Atg8 protein family for efficient internalization and degradation. Secondly, APP binds directly to beclin 1 and recruits the clathrin mediated internalization machinery, mainly AP2 via lipidated LC3. This leads to APP internalization and degradation without A peptide

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generation. Thirdly, ECHO virus 7 and WSSV use the autophagic machinery for their clathrin dependent internalization. WSSV recruits the Atg8 family member GABARAP to its entry sites for this purpose. Fourthly, Ebola virus recruits Ankfy1 via LC3 binding for internalization via

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macropinocytosis.