Antigen Presentation Autophagy and Its Role in MHC-Mediated

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Autophagy and Its Role in MHC-Mediated Antigen Presentation Victoria L. Crotzer and Janice S. Blum This information is current as J Immunol 2009; 182:3335-3341; ; of October 2, 2021. doi: 10.4049/jimmunol.0803458 http://www.jimmunol.org/content/182/6/3335

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Autophagy and Its Role in MHC-Mediated Antigen Presentation1 Victoria L. Crotzer and Janice S. Blum2

Intracellular degradation by autophagy plays a role in hydrolase sorting in yeast (6). Another pathway, xenophagy, in- the maintenance of cellular homeostasis under normal volves the use of the autophagic machinery for the degradation conditions and during periods of cellular stress. Auto- of intracellular pathogens (7). Macroautophagy and CMA have phagy has also been implicated in several other cellular been implicated in the presentation of Ag by MHC molecules processes including immune recognition and respon- and, thus, are the major focus of this review. siveness. More specifically, autophagy has been identi- Macroautophagy is also a constitutively active cellular process modulating the degradation of long-lived proteins and or-

fied as a route by which cytoplasmic and nuclear Ag are Downloaded from ganelles (1). During short periods of nutrient deprivation, this delivered to MHC class II molecules for presentation to ؉ pathway is rapidly induced as a mechanism to salvage amino CD4 T cells. Autophagy has also recently been impli- acids. The absence of growth factors or amino acids prevents cated in MHC class I cross-presentation of tumor Ag -؉ signaling through PI3K from activating the autophagy regula and the activation of CD8 T cells. This review dis- tory molecule known as mammalian target of rapamycin cusses the role of autophagy in modulating MHC class (mTOR), and thus, macroautophagy is induced (8). In macro- I and class II Ag presentation as well as its implication autophagy, portions of the cytoplasm are sequestered into dou- http://www.jimmunol.org/ in regulating autoimmunity and tolerance, tumor ble-membrane structures known as autophagosomes. The immunity, and host defense against intracellular formation of the autophagosome requires a number of autoph- pathogens. The Journal of Immunology, 2009, 182: agy-related gene (Atg) products that have been well-character- 3335–3341. ized in yeast (6) and are conserved in mammals; the specific details of autophagosome formation have been reviewed extensively elsewhere (2, 8). Briefly, the Atg12-Atg5-Atg16 complex, utophagy is one mechanism that cells use to degrade in conjunction with Atg9, mediates the induction of autopha- cytoplasmic proteins and organelles during the main- gosome formation. During this process, LC3 (Atg8) is conju- by guest on October 2, 2021 A tenance of cellular homeostasis (1). There are multi- gated to phosphatidylethanolamine with the assistance of the ple pathways of autophagy. In microautophagy, small portions Atg4, Atg7, and Atg3 molecules and incorporated into the au- of the cytosol are internalized via lysosomal membrane invagi- tophagosomal membrane. This membrane next expands and nations, and proteins are continuously degraded in the lumen engulfs portions of the cytosol, including entire organelles. of this organelle even under resting conditions (2). Microauto- Upon formation of the autophagosome, the Atg12-Atg5-Atg16 phagy is up-regulated under conditions of cellular stress such as complex dissociates from this structure while LC3 (Atg8)-phos- nutrient deprivation, but studies of this pathway have been lim- phatidylethanolamine remains in the autophagic lumen as an ited to yeast and cell-free systems (3, 4). In mammalian cells, autophagosomal marker. The outer membrane of the autopha- constitutively active autophagy pathways can also be up-regu- gosome fuses with the lysosomal membrane, forming an autoly- lated as a mechanism to salvage amino acids during periods of sosome within which the single membrane structure, or auto- cellular stress. Several types of selective autophagy have been phagic body, is degraded by lysosomal esterases, lipases, and identified in mammalian cells including macroautophagy and proteases. chaperone-mediated autophagy (CMA)3 (Fig. 1), as well as During periods of prolonged cellular stress, bulk autophagy pexophagy and mitophagy (5). A related constitutive biosyn- declines, and the process of CMA is up-regulated. In CMA, spe- thetic pathway, cytoplasm-to-vacuole targeting, is involved in cific cytosolic proteins displaying homologous pentapeptide

Department of Microbiology and Immunology and the Walther Oncology Center, Indi- 3 Abbreviations used in this paper: CMA, chaperone-mediated autophagy; ALIS, ag- ana University School of Medicine and the Walther Cancer Institute, Indianapolis, IN gresome-like induced structures; Atg, autophagy-related gene; cTEC, cortical thymic 46202 epithelial cell; DC, dendritic cell; EBNA, EBV nuclear Ag; ER, endoplasmic reticulum; GAD, glutamic acid decarboxylase; hsc70, heat shock cognate 70-kDa protein; Ii, Received for publication December 19, 2008. Accepted for publication January 21, 2009. invariant chain; LAMP, lysosome-associated membrane protein; 3-MA, 3-methylad- The costs of publication of this article were defrayed in part by the payment of page charges. enine; MP1, influenza matrix protein 1; mTOR, mammalian target of rapamycin; This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. siRNA, small interfering RNA. Section 1734 solely to indicate this fact. Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 1 V.L.C. is supported by a Career Development Award from the Melanoma Research Foundation. National Institutes of Health AI49589 supported the effort of J.S.B. 2 Address correspondence and reprint requests to Dr. Janice S. Blum, Medical Science Building Room 420, 635 Barnhill Drive, Indianapolis, Indiana, 46202-5120. E-mail address: [email protected] www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803458 3336 BRIEF REVIEWS: AUTOPHAGY AND ANTIGEN PRESENTATION

class I presentation pathway is still under investigation, although several routes have recently been proposed. These pathways have been described extensively elsewhere (11–13); thus, only a brief summary of the current models of class I-mediated cross-presentation is offered here. Studies have suggested that MHC class I molecules may acquire peptides derived from exogenous Ag internalized into phagosomes in a TAP-dependent or TAP-independent manner. In the TAP-dependent phagosome-to-cytosol-to-phagosome pathway, Ag is transported to the cytosol by a yet unknown mechanism, possibly Sec61, and degraded by the proteasome. Peptides are then reimported into the phagosome, FIGURE 1. Pathways of autophagy in MHC class II-mediated Ag presen- which has acquired TAP as well as other ER proteins such as tation. In macroautophagy, the cytoplasm is sequestered into double-mem- MHC class I. The phagosome-to-cytosol pathway calls for pep- brane structures known as autophagosomes, which fuse with lysosomes. In tides generated in the cytosol by the proteasome to be directly CMA, specific cytosolic proteins are transported into lysosomes via a molecular transported to MHC class I molecules in the ER via TAP. An- chaperone/receptor complex composed of hsc70 and LAMP-2A. other possible mechanism of class I-restricted cross-presentation may involve the ER-associated degradation pathway. Here, exogenous Ag internalized in endosomes is transported into the Downloaded from motifs bind to a molecular chaperone complex composed of ER, translocated for degradation in the cytosol by the protea- multiple heat shock proteins including the heat shock cognate some, and finally transported back into the ER by TAP. It has 70-kDa protein (hsc70) (reviewed in Ref. 9). This molecular also been reported that DC may acquire exogenous peptides ϩ chaperone complex transports the substrate protein to the lyso- from other cells via gap junctions to cross-prime naive CD8 T somal membrane, where it associates with an isoform of the ly- cells. Lastly, in the vacuolar pathway, MHC class I molecules http://www.jimmunol.org/ sosome-associated membrane protein (LAMP) 2A, which func- may acquire peptides that are generated in phagosomes by cys- tions as part of the lysosomal receptor for CMA. The teine proteases such as cathepsin S in a TAP-independent man- cytoplasmic domain of LAMP-2A serves as a potential docking ner. The mechanisms that potentiate MHC class I peptide load- site for the molecular chaperone-substrate complex, and chap- ing in phagosomes via this latter pathway have not yet been erone-mediated substrate unfolding likely enables proteins to determined. bind and translocate across the lysosomal membrane. Lysoso- Autophagy has recently been proposed as another possible mal hsc70 assists with the transport of substrate proteins into mechanism for the MHC class I-mediated cross-presentation of the organelle lumen, where these molecules are degraded by exogenous Ag with results pertinent to tumor Ag (14). In this mature acidic proteases. study, the authors induced macroautophagy in HEK 293T cells by guest on October 2, 2021 expressing a model Ag OVA by starvation or treatment with the Autophagy and MHC class I Ag presentation compound rapamycin, which inhibits the mTOR molecule In the conventional paradigm, MHC class I molecules are re- (15). Following these treatments in vitro, enhanced cross-pre- ϩ stricted to surveying the cytosol for endogenous Ag from vi- sentation of OVA to epitope-specific CD8 T cells was ob- ϩ ruses, tumors, or self-proteins for presentation to CD8 T cells served. Conversely, the treatment of the OVA-expressing HEK (reviewed in Ref. 10). These endogenous Ag are degraded into 293T cells with 3-methyladenine (3-MA), an inhibitor of PI3K peptide fragments by cytosolic proteases such as the proteasome and macroautophagy (16), significantly reduced OVA cross- and transported via a heterodimeric complex composed of presentation in vitro. These authors also used rapamycin, TAP1 and TAP2 into the endoplasmic reticulum (ER). Once in 3-MA, and another PI3K inhibitor, wortmannin (17), to mod- the ER, peptides of the appropriate length (8–10 aa) bind nas- ulate macroautophagy in melanoma cells and then measured ␤ cent MHC class I H chains and 2-microglobulin and then the MHC class I-restricted cross-presentation of the tumor Ag transit through the secretory pathway to the cell surface for rec- gp100 in vivo. Similar to the results with the OVA-expressing ϩ ognition by CD8 T cells. A direct role for autophagy in the HEK 293T cells, induction of macroautophagy enhanced the conventional pathway for MHC class I presentation has not cross-presentation of gp100 whereas inhibition of macroauto- been firmly established, yet recent studies discussed below dem- phagy reduced the cross-presentation of this Ag to epitope-spe- ϩ onstrate potential roles for autophagy in regulating class I-me- cific CD8 T cells. Macroautophagy was also specifically in- diated cross-presentation of exogenous Ag. hibited through the use of small interfering RNA (siRNA) During the process of cross-presentation, bone marrow-de- against the autophagy gene Atg6/beclin-1 in melanoma cells, rived APC such as dendritic cells (DC) internalize and degrade and a significant reduction in the in vivo MHC class I-mediated Ag from the extracellular environment and display the resulting cross-presentation of gp100 was again observed. peptides in association with MHC class I molecules on their cell These results support a potential role for macroautophagy in ϩ surfaces (11). The stimulation of naive CD8 T cells by these the MHC class I-restricted cross-presentation of tumor Ag; yet peptide-MHC class I complexes is known as cross-priming. further mechanistic studies to define the role of autophagy The class I-restricted cross-presentation pathway plays an im- pathways in class I-mediated Ag presentation are needed. The portant role in the immune surveillance of infected cells as well induction of macroautophagy by rapamycin in the OVA-ex- as tumors and the subsequent development of an appropriate pressing HEK 293T cells as well as the melanoma cells leads to cytotoxic T cell response to these pathogens or malignancies. the conversion of LC3-I to its LC3-II form, suggesting that the Precisely how peptides from exogenous Ag access the MHC MHC class I-restricted cross-presentation of OVA and gp100 The Journal of Immunology 3337

FIGURE 2. Autophagy as a potential mechanism for MHC class I-mediated presentation of tumor Ag. Macroauto- phagy can be induced in tumor cells undergoing cellular stress as a result of nutrient depletion or chemotherapy. Autophago- somes containing tumor Ag may be released into the extracellular environment where they are internalized by DC. How and where tumor Ag in autophagosomes intersect with MHC class I molecules in DC is unknown. DC present tumor peptides in the context of class I for the activation of CD8ϩ T cells. may require the initiation and formation of autophagosomes. MHC class II complexes consist of ␣ and ␤ subunits, which are Yet, treatment of cells with NH4Cl, which blocks the acidifica- first assembled in the ER with the chaperone molecule invariant tion of lysosomes and prevents their fusion with autophago- chain (Ii) (28, 29). The cytoplasmic tail of Ii contains a motif somes, enhances class I-mediated cross-presentation. These re- that targets the Ii-MHC class II complexes to endosomal/lyso- sults suggest that the degradation of the contents of somal compartments where acidic proteases degrade Ii to a autolysosomes during the late phases of autophagy hinders small fragment known as the class II-associated invariant chain cross-presentation, perhaps by destroying the Ag that serve as peptide or CLIP, which remains associated with the MHC class the sources of peptide epitopes for MHC class I molecules. In II peptide binding groove (30, 31). Ag delivered into the endo- Downloaded from support of this, Li et al. show that purified autophagosomes somal/lysosomal network via receptor-mediated or fluid phase from OVA-expressing HEK 293T cells or melanoma cells serve endocytosis are also exposed to proteases and denaturing reac- as a source of Ag for DC to promote cross-presentation of OVA tions, yielding peptide ligands for class II molecules (32). CLIP ϩ or gp100 to the appropriate epitope-specific CD8 T cells (14). removal and the capture of antigenic peptides by MHC class II Although the authors demonstrate that autophagosomes may proteins is catalyzed by the MHC-encoded molecule HLA-DM function as efficient carriers of Ag from donor cells, how and (33–35) and occurs in a highly specialized organelle resembling http://www.jimmunol.org/ where these structures and their antigenic contents intersect a late endosome/lysosome known as the MIIC (36). The result- with MHC class I molecules have yet to be determined (Fig. 2). ant peptide-MHC class II complexes are ultimately trafficked to ϩ Further insights into the specific mechanisms by which auto- the cell surface for immune surveillance by CD4 T cells. phagy plays a role in the presentation of tumor Ag by MHC Similar to the MHC class I-mediated cross-presentation of ϩ class I molecules may lead to the development of new therapeu- exogenous Ag to CD8 T cells, recent studies have begun to tic strategies for the treatment of cancer. reveal how cytoplasmic and nuclear Ag gain access to MHC In addition to its role in tumor immunity, autophagy may class II molecules in lysosomal compartments for the presenta- ϩ ϩ also contribute to the CD8 T cell response to intracellular tion of peptides to CD4 T cells (37, 38). Multiple pathways pathogens such as bacteria, parasites, and viruses. For example, have been shown to contribute to the MHC class II-mediated by guest on October 2, 2021 autophagy is required for the clearance of several pathogens (re- presentation of cytoplasmic and nuclear viral, tumor, and self viewed in Refs. 18–20), including the parasite Toxoplasma gon- Ag in both professional and nonprofessional APC. These path- dii, which is localized in autophagosomes in infected macro- ways include CMA, macroautophagy, a TAP-dependent path- ϩ phages (21, 22). Because CD8 T cells are essential for way, and intercellular Ag transfer. The findings of these studies protective immunity from T. gondii (23), it is possible that pep- have been reviewed extensively elsewhere (39–42). Thus, in tides derived from T. gondii access MHC class I molecules this review only a brief summary of these pathways for MHC through autophagy. Several pathogens have been localized in class II-mediated endogenous Ag presentation is offered autophagosomes that contain ER proteins (24), and perhaps (Fig. 3). components of the MHC class I presentation pathway such as Recently, our laboratory demonstrated a role for CMA in class I molecules or TAP may also be found in autophagosomes. regulating cytoplasmic Ag presentation by MHC class II mol- Furthermore, during periods of cellular stress, aggresome-like ecules by modulating the cellular levels of two components of induced structures (ALIS) containing polyubiquitylated pro- the CMA pathway, LAMP-2A and hsc70, in human B cells ex- teins are formed and serve as substrates for autophagy (25). pressing cytoplasmic Ag glutamic acid decarboxylase (GAD) or ALIS can form in DC (designated DALIS) experiencing cellular a mutant form of human Ig ␬ L chain, designated SMA (37). stress, perhaps as a result of an infection by a pathogen, and the Expression of antisense cDNA for LAMP-2 reduced the MHC clearance of these DALIS by autophagy has been proposed as a class II-restricted presentation of GAD, whereas overexpression source of peptides for MHC class I molecules (26). of the LAMP-2A isoform resulted in an increased presentation of epitopes from both GAD and SMA. In B cells, SMA, one of Autophagy and MHC class II Ag presentation the causative agents of L chain amyloidosis, fails to fold properly Traditionally, MHC class II molecules present antigenic pep- after synthesis and is immediately translocated out of the ER to ϩ tides derived from exogenous proteins to CD4 T cells (27). the cytosol (43), suggesting that CMA may contribute to the MHC class II proteins are constitutively expressed on the sur- immune recognition of misfolded ER proteins. Stress-induced face of a number of professional APC such as DC, B cells, and autophagy pathways also appear to play a role in the presenta- macrophages, as well as both cortical and medullary thymic ep- tion of multiple cytoplasmic and nuclear Ag. Human B cells in ithelial cells (cTEC and mTEC, respectively). Treatment with which autophagy is induced by serum starvation display an in- an inflammatory signal such as IFN-␥ can induce MHC class II crease in those MHC class II-associated peptides derived from expression on the surfaces of nonprofessional APC such as en- intracellular proteins as measured by mass spectrometry (44). dothelial cells, fibroblasts, epithelial cells, and many tumors. Studies using the PI3K inhibitors 3-MA and wortmannin or 3338 BRIEF REVIEWS: AUTOPHAGY AND ANTIGEN PRESENTATION

FIGURE 3. Autophagy pathways in the MHC class-mediated presentation of cytoplasmic and nuclear Ag. Approximately 10–25% of MHC class II molecules display peptides from nuclear and cytoplasmic Ag likely accessing autophagy to reach class II. Left, Cytoplasmic Ag are first processed by cytosolic proteases and then use CMA to access lysosomes. Middle, These antigenic fragments intersect with MHC class II molecules in a mature endosomal/lysosomal compartment known as the MIIC before presentation to CD4ϩ T cells. Right, Cyto- plasmic or nuclear Ag are sequestered into double- membrane structures known as autophagosomes that continuously fuse with mature endosomes such as the MIIC and lysosomes, thus allowing Ag to associate with MHC class II molecules. Further processing of the cytoplasmic or nuclear Ag by lysosomal proteases may occur before binding to class ϩ II molecules and presentation to CD4 T cells. Downloaded from

siRNA targeting the Atg12 gene to block macroautophagy Autophagy and the regulation of immune responses http://www.jimmunol.org/ demonstrated that multiple epitopes from cytoplasmic and nu- Autophagy pathways may be induced or altered during infec- clear proteins are dependent on this pathway for their presen- tion and immunity as cellular stress responses are initiated. In tation by MHC class II molecules. These epitopes include the vitro studies have demonstrated recognition of viral, self Ag, cytosolic and nuclear versions of the bacterial protein neomycin and tumor Ag dependent upon autophagy in the context of (45, 46), mucin-1 (47), and the EBV nuclear Ag (EBNA) 1 MHC class II Ag presentation. A role for autophagy in the (48). A recent report by Munz and colleagues demonstrates that cross-presentation of tumor Ag via MHC class I molecules has in B cells and DCs, autophagosomes are constitutively formed also been reported both in vitro and in vivo (14). Less is known and continuously fuse with MIIC (38). Furthermore, when the ϩ ϩ

about whether autophagy influences either CD4 or CD8 T by guest on October 2, 2021 influenza matrix protein 1 (MP1) was targeted to autophago- cell responses to bacterial pathogens. A recent report on auto- somes by fusion with LC3 (Atg8), an increase in the MHC class ϩ phagy in the thymus provides a clue as to the importance of this II-restricted presentation of this epitope to CD4 T cells was process in the presentation of self Ag and its impact on the in- ϩ observed. duction and maintenance of CD4 T cell tolerance (55). The Two additional pathways have been shown to contribute potential role of autophagy in regulating immune responses to to the MHC class II-mediated presentation of endogenous pathogens, tumors, and self Ag has been reviewed extensively viral Ag in B cells and DC, including a TAP-dependent path- elsewhere (18, 41, 56, 57), with highlights of recent studies dis- way and intercellular Ag transfer. Both the proteasome and cussed here. TAP were shown to be required for the generation of two MHC class II-restricted epitopes encoded within two dis- tinct transmembrane glycoproteins from influenza virus us- Intracellular pathogens ing murine B cells and DC (49). These results differ from Studies have begun to examine the role of autophagy in a host several reports suggesting that TAP plays no role in the pre- cell’s defense against intracellular pathogens such as viruses and sentation of cytoplasmic Ag to MHC class II molecules (37, bacteria, although the precise molecular steps involving auto- 46, 50–53); thus, further studies regarding this transport phagy or autophagy-linked gene products are still poorly under- pathway in specialized immune cells such as DC are clearly stood (reviewed in Refs. 41 and 58). Autophagy limits the virus- needed. In the second pathway, two EBV nuclear Ag, EBNA induced encephalitis observed during Sindbis virus infection 2 and EBNA 3C, were shown to gain access to the MHC through the interaction of the Atg6 gene Beclin 1 with Bcl-2 class II pathway via intercellular Ag transfer (54). Treatment (59). Additionally, during the process of xenophagy, autopha- of EBV-transformed human B cells with 3-MA to block gosomes engulf and destroy viruses (7). For example, HSV-1 is macroautophagy failed to inhibit the MHC class II-re- degraded within autophagosomes in a dsRNA-activated protein stricted presentation of these endogenous viral Ag. Whether kinase-dependent manner (60). Autophagy has recently been intact viral Ag or antigenic fragments complexed with other shown to play a role in the innate immune response to vesicular proteins or within exosomes are transferred to recipient APC stomatitis virus (61). In plasmacytoid DC, autophagy facilitates for MHC class II-mediated presentation is unknown. How- the transport of vesicular stomatitis virus replication interme- ever, Ag processing by lysosomal proteases within the recip- diates from the cytosol to lysosomes potentially to promote ient cell is required for the class II-restricted presentation of TLR7 engagement. Activation of TLR7 in DC results in the EBNA 2 and EBNA 3C viral Ag, strongly supporting inter- induction of important mediators of antiviral immunity such as cellular or cross-presentation. IFN-␣ and IL-12. Plasmacytoid DC also recognize viruses The Journal of Immunology 3339 through TLR9 localized in endosomes, and signaling through induction of autophagy within a tumor cell, the formation of TLR9 recruits and activates the transcription factor IFN re- autophagosomes might provide a source of tumor Ag that could sponse factor 7, which is essential for the production of type I intersect with MHC class II molecules upon fusion of the au- IFNs (62). Using rapamycin and siRNA against mTOR, Cao et tophagosomes with lysosomes. al. recently demonstrated that virus-induced TLR9-dependent signaling through the mTOR pathway is also critical for the production of type I IFNs in plasmacytoid DC as well as bone- Autoimmunity and tolerance marrow derived DC and macrophages (63). Whether signaling Autophagy appears to also play a role in the breakdown of tol- through mTOR following virus-induced activation of TLR9 erance and the development of autoimmunity (reviewed in affects the autophagy pathway during an innate antiviral im- Refs. 41 and 69). During autophagy, contents of the cytosol mune response has yet to be investigated. Lastly, as discussed and nucleus, including potential self-Ag, are degraded in lyso- above, the MHC class II-mediated presentation of the EBV Ag somal compartments where MHC class II molecules reside. EBNA1 by autophagy suggests that this pathway may also play Thus, alterations in autophagy pathways may lead to a break- a role in the adaptive immune response to viruses (48). The down in tolerance or to insufficient tolerance induction and the demonstration that targeting influenza MP1 to autophago- development of autoimmunity. A recent report by Nedjic et al. somes enhanced MHC class II presentation of the MP1 epitope demonstrated that autophagy plays a key role in the generation (38) may suggest that in virally infected cells, fusion of auto- and maintenance of tolerance in the thymus (55). In this study, phagosomes containing viral particles with lysosomes could

autophagosomes were not readily detected in murine peripheral Downloaded from promote viral Ag association with class II for presentation to ϩ APC, yet cTEC, which express MHC class II molecules, were CD4 T cells. found to display high levels of constitutive macroautophagy. Host cells also use autophagy pathways in their defense The authors postulated that macroautophagy within cTEC against bacteria and parasites. Autophagy limits bacterial repli- may favor the presentation of a broader spectrum of self Ag via cation by enveloping free bacteria such as group A Streptococcus Ϫ/Ϫ class II on these cells. Using thymi from Atg5 mice, the au- in autophagosomes for delivery to lysosomes (64) or by target- thors evaluated the positive and negative selection of several http://www.jimmunol.org/ ing phagosomes containing bacteria such as Mycobacterium tu- MHC class II-restricted transgenic TCRs and found that the berculosis for fusion with lysosomes (65, 66). The innate im- loss of Atg5 altered the selection of some, but not all, TCRs. mune response initiated as a result of pathogenic infections also These results suggested that in the absence of autophagy, the influences autophagy pathways. For example, signaling through composition of peptide-MHC class II complexes on the surface TLR7 during infection of macrophages with Mycobacterium bo- of cTEC was altered, thus skewing the T cell repertoire that was vis bacilli Calmette-Guerin induces macroautophagy, resulting in the elimination of the intracellular bacteria (67). Further- positively selected. Additionally, the lack of autophagy may also more, cytokine expression may modulate macroautophagy to prevent MHC class II presentation of certain tissue-specific Ag limit pathogen replication. Type II IFN enhances the degrada- in medullary thymic epithelial cells, thus impairing negative se- by guest on October 2, 2021 tion of M. tuberculosis and Rickettsia conorii in infected cells (65, lection and allowing autoreactive T cells to exit into the periph- 66), and the TNF family member CD40 ligand induces mac- ery. In support of this, the authors observed severe colitis and inflammation of multiple organs in athymic mice that received roautophagy to facilitate the fusion of phagosomes containing Ϫ/Ϫ T. gondii with lysosomes during parasite infection (21). Finally, thymi from Atg5 mice. Studies of T cells deficient in Atg5 although the role of autophagy in the adaptive immune re- revealed defects in cellular proliferation and survival (70), sug- sponse to bacteria and parasites has not yet been carefully ex- gesting further analysis of the role of this gene product in reg- amined, it is possible that in infected cells the fusion of auto- ulating T cell responses as well as the thymic microenvironment phagosomes containing intracellular pathogens with lysosomes is needed. could be a mechanism by which bacterial or parasitic Ag asso- In humans, recent studies have identified ATG16L1 (auto- ϩ ciate with MHC class II molecules for presentation to CD4 T phagy-related 16-like 1), which is involved in autophagosome cells. formation, and IRGM GTPase, which stimulates autophagy, as susceptibility loci for Crohn’s disease (71–74). It has been pro- Cancer posed that mutations in these genes may lead to defective mac- Similar to its role in infectious disease, autophagy is believed to roautophagy, thus impairing the innate immune response to contribute to both cancer development and tumor suppression bacterial infection and increasing the bacterial load in the gut, (57). Autophagy may be induced during the later stages of on- resulting in enhanced mucosal inflammation and the develop- cogenesis as the rapid proliferation of the tumor depletes critical ment of disease. A recent study using ATG16L1-deficient mice nutrients, thus suppressing further tumor growth. Additionally, demonstrated an increase in the production of the inflamma- following chemotherapy, tumor cells may initiate autophagy as tory cytokines IL-1␤ and IL-18 and in the susceptibility to co- Ϫ/Ϫ a protective measure to recycle nutrients or remove damaged litis (75). Alternatively, as the data from the Atg5 mice cellular components. For example, the induction of macroau- would suggest, inhibition of autophagy pathways might also tophagy increases the survival of breast cancer cells undergoing lead to the failure to induce tolerance to mucosal self Ag. Lastly, treatment with 4-hydroxytamoxifen (68). Yet, how the modu- the tissue damage associated with certain autoimmune diseases lation of autophagy pathways in tumor cells influences immune such as systemic lupus erythematosus has been attributed to the recognition and clearance has not yet been thoroughly investi- defective clearance of apoptotic cells. Macroautophagy plays a gated. As discussed above, one recent report suggests autophagy role in the removal of these apoptotic cell corpses, and the im- as a potential mechanism for the cross-presentation of tumor Ag pairment of this process may result in the buildup of necrotic onto MHC class I molecules. It is also possible that during the material in tissues and, thus, inflammation. 3340 BRIEF REVIEWS: AUTOPHAGY AND ANTIGEN PRESENTATION

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