The functions of autophagy at the tumour‐immune interface

Abstract Autophagy is frequently induced in the hypoxic tumour microenvironment. Accumulating evidence reveals important functions of autophagy at the tumour‐immune interface. Herein, we propose an update on the roles of autophagy in modulating tumour immunity. Autophagy promotes adaptive resistance of established tumours to the cytotoxic effects of natural killer cells (NKs), macrophages and effector T cells. Increased autophagic flux in tumours dampen their immunogenicity and inhibits the expansion of cytotoxic T lymphocytes (CTLs) by suppressing the activation of STING type I interferon signalling (IFN‐I) innate immune sensing pathway. Autophagy in suppressive tumour‐infiltrating immune subsets maintains their survival through metabolic remodelling. On the other hand, autophagy is involved in the antigen processing and presentation process, which is essential for anti‐tumour immune responses. Genetic deletion of autophagy induces spontaneous tumours in some models. Thus, the role of autophagy is context‐dependent. In summary, our review has revealed the dichotomous roles of autophagy in modulating tumour immunity. Broad targeting of autophagy may not yield maximal benefits. The characterization of specific genes regulating tumour immunogenicity and innovation in targeted delivery of autophagy inhibitors into certain tumours are among the most urgent tasks to sensitize cold cancers to immunotherapy.


| BACKG ROU N D
Autophagy serves as an evolutionarily conserved physiological phenomenon to maintain cellular homeostasis and survival during nutrient deprivation. The initiation of autophagic response is briefly presented as the encapsulation of excessive or damaged cellular components and organelles into autophagosome leading to enzymatic degradation. 1,2 According to the various delivering routes and contents to lysosomes, autophagy is generally categorized into macroautophagy (predominant form generally termed as autophagy), microautophagy and chaperon-mediated autophagy. 3 Autophagy is also frequently altered under pathological circumstances, namely hypoxia, endoplasmic reticulum (ER) stress, nutrient deficiency, radiation and chemotherapy. [4][5][6] Aside from its direct effect on cancer cell response to environmental challenges, recent studies show that autophagy in cancer cells regulates tumour-immune interactions, depending upon the context of cancer types, metabolic alterations in the tumour microenvironment (TME) and the stage of cancers. 7 Genetic evidence showed that autophagy is a critical mechanism suppressing tumour initiation, 8 however, in established tumours autophagy contributes to adaptive resistance. 9 Of note, mitophagy, another form of autophagy, plays a similar role in regulating tumour development by adjusting tumour immune response. 10 In this review, we seek to summarize recent evidence characterizing the functions of autophagy, including mitophagy, in regulating tumour-immune interactions (Figures 1-3; Table 1).

| AUTOPHAGY IN E S TAB LIS HED TUMOUR S PROMOTE S E VA S ION FROM INNATE AND ADAP TIVE IMMUNE SURVEILL AN CE
Autophagy could directly or indirectly exert its effect on the innate immunity mediated by natural killer (NK) cells, dendritic cells (DCs) and macrophage population. First, autophagy of tumour cells promotes adaptive resistance to NK-induced tumour lysis. NK cells, which are considered as the first-line defence against tumours, releasing perforin, and granzyme B for the lysis of tumour cells. 11,12 Its anti-cancer role has been validated in malignancies, such as gastric cancer 13 and lung cancer. 14 By exploiting the in vivo and in vitro breast cancer models, Baginska et al observed that the autophagy provoked by the hypoxic TME is involved in the degradation of granzyme B originated from NK into cancer cells, thus counteracting the apoptotic cell death effect induced by NK cells. 12 Besides, several studies also suggest additional mechanisms that contribute to the low tumour immunosurveillance and cytotoxicity of NK cells for cancers. Gap junctions (GJs) are interacting channels that mediate the exchange of the small molecules between cells composed by connexin subunits, among which Connexin 43 (Cx43) is uncovered as the major GJ protein located at the immunological synapse and bridging the interplay between immune cells and cancer cells. 15 Hypoxia-induced autophagic flux results in the degradation of Cx43 in melanoma cells and impairs the cytotoxic effects of NK cells upon cancer cells. In agreement, elevated Cx43 expression levels in tumour cells are beneficial to enhance the efficacy of NK-based immunotherapy. 15 Inositol 1,4,5-trisphosphate receptor, type 1 (ITPR1), as one ligand-gated channel of ion for managing calcium release from the endoplasmic reticulum, is reported to be able to induce autophagy. 16 Messai's study regarding clear cell renal cell carcinomas (CCRCC) indicated that the elevated expression of ITPR1 evoked by HIF-2α initiated the autophagic degradation of granzyme B and abolished the NK-induced killing effect on tumour cells. In agreement with that, they implanted the tumours in mice and observed reduced tumour growth by inhibiting ITPR, while the depletion of NK cells reverted the tumour suppression. 17 As another line of evidence of autophagy-mediated immune resistance, depletion of autophagy-promoting Beclin 1 (BECN1) leads to increased intensity of chemokine (C-C motif) ligand 5 (CCL5) expression within melanoma cells and redirects massive NK cells into the tumour microenvironment, thus leading to tumour suppression. 18 Macrophages may also exert innate immune surveillance in the TME through their phagocytic functions. [19][20][21] A glioblastoma study employing a combinatorial treatment to target both VEGF and CD47, the latter of which inhibited the phagocytic effect of macrophages, revealed that it could trigger autophagy of cancer cells which attenuated the phagocytosis and cytotoxicity of macrophage population. Inhibition of various signalling pathways, including Akt/mTOR and Erk, was responsible for the enhanced autophagy. 21 The same group also demonstrated that the combination of anti-CD47 therapy with autophagy inhibitor would robustly improve the therapeutic efficacy against non-small cell lung cancer (NSCLC).These results suggest that autophagy originated from tumour cells could impede the phagocytic function of macrophages. 22 Zhang et al found that autophagy occurring in glioblastoma cells could mitigate the immunotherapeutic efficacy of anti-CD47-SIRPα treatment, displaying as the reduced macrophage-derived phagocytosis and subsequent attenuation of CD8 + T-cell cytotoxicity. 23 Notably, macrophages may be regulated by tumour cell-released autophagosomes (TRAPs) and affect the cytotoxic T lymphocytes (CTLs). 24 TRAPs are a type of double-membrane vesicles released into the TME by tumour cells, which escape from the lysosome fusion stage of classical autophagy. 25 Wen and colleagues uncovered that within several tumour models, the TRAPs could skew macrophages into M2-phenotype with higher levels of PD-L1 and IL-10 via Toll-like receptor 4 (TLR4)-MyD88-p38-STAT3 pathway, therefore resulting in suppression of CTL function and reduced IFNγ secretion. 24 Moreover, tumour-associated autophagy also contributes to evasion from adaptive immunity. For example, the response rate of head and neck squamous cell carcinoma (HNSCC) to immunotherapy remains less than 15%, for which low immunogenicity and a poor infiltration of CTLs were indicated as the possible reason. 26,27 Type I interferon (IFN-I) signalling promotes anti-tumour effects by mediating the recruitment and maturation of antigen-presenting cells (APCs).
Stimulator of IFN genes (STING) is a pivotal adaptor protein that could activate the IFN-I pathway. 28,29 Nonetheless, STING is frequently inhibited in TME, contributing to tumour escape from innate immune sensing. Recent studies identified previously unknown functions of oncogenes in suppressing the STING-IFN-I innate immune sensing pathway. 30,31 Specifically, SOX2, previously known as a cancer stemness gene, was correlated with immunosuppression. 30 In addition, the results of in vivo experiment suggested that SOX2expressing tumours contained lower numbers of CD8 + CTLs and that those infiltrating T cells expressed higher levels of PD-1 than SOX2negative tumours. 30 HPV + HNSCC is driven by a distinct aetiology, with different immune infiltration patterns from HPVtumours.
Interestingly, HPV + HNSCCs contain less T-cell receptor richness, in contrast to its usually heavy immune infiltration. 34 IFN-I is essential for tumour-specific CTL expansion. A recent study showed that HPV16 E7 could contribute to the autophagic degradation of STING by binding to NLRX1, which was shown to promote autophagosome formation. 5,31,35,36 NLRX1 deficiency in the tumour cells promoted CD8 + CTL expansion located in the tumour-draining lymph nodes and reduces CTL exhaustion in the TME. 31 In agreement, additional studies also found that high-risk HPV subtypes utilize a number of strategies to antagonize IFN-I induction [37][38][39] ; Gariglio and colleagues observed that HPV E7 could attenuate the IFN-I activation in HPVtransformed cells via epigenetic silencing of sensor genes including RIG-I, cGAS and STING in an SUV39H1-dependent manner. 37 NLRX1 has an LC3-interacting region and can directly interact with LC3. Such interaction underpins an NLRX1-mediated mitophagy process. Depletion of NLRX1 promotes mitochondria-derived reactive oxygen species, which arguably amplifies the production of Th1 cytokines. 40 The role of autophagy in inhibiting IFN-I was also corroborated using a transgenic FIP200 (FAK family-interacting protein of 200 kD)-deficient mouse model. FIP200 is an essential autophagy gene, in the absence of which mammary tumorigenesis is suppressed. The study found that inhibition of autophagy promoted the activation of IFN-I signalling as well as its downstream chemokines such as CXCL10, subsequently inducing CD8 + CTL expansion in the TME. 41 Of interest, Yamamoto and colleagues recently reported that autophagy is responsible for the degradation of MHC-I in pancreatic ductal adenocarcinoma by employing the autophagy cargo receptor NBR1, resulting in the tumour immune evasion. 42 Thus, modulating selective autophagy represents a non-tapped approach to fine-tune host immune responses.
Additional evidence implies that autophagy is also responsible for tumour immune escape by stimulating signal transducer and activator of transcription 3 (STAT3) signalling, an oncogenic pathway.
The STAT3 pathway has been an important link between tumour and immune cells. 7,43 Wang et al reported that STAT3 activation occurring in tumour cells could significantly reduce the production of proinflammatory cytokines and chemokines critical for APC maturation and its recruitment to the tumour bed. 44 Autophagy has been shown to increase STAT3 phosphorylation in multiple tumour models. [45][46][47] Autophagy may also inhibit adaptive immunity by dampening the immunogenic cell death (ICD)-induced immune killing. ICD can be triggered by several anti-cancer treatments such as chemotherapy, radiotherapy 48  (ATP). [51][52][53] These are pivotal to the proper processing of antigen by APCs, and these molecules, including CRT, HMGB1 and ATP were defined as damage-associated molecular patterns (DAMPs). 54 Garg et al reported that by genetically blocking autophagy in the tumour model under Hyp-PDT, an increase in CRT and ICD-caused immune reaction was detected. This was elucidated as the up-regulation of IL6-producing mature DCs and CTLs along with IFNγ. 50 In addition to tumour-intrinsic autophagy, immune cell-inherent autophagy may also deliver resistance to immune killing. Myeloidderived suppressor cells (MDSCs) and regulatory T cells (Tregs) are the dominant subsets in the TME to promote tumour immune escape. 55,56 HMGB1-induced autophagy was found essential for maintaining the survival of MDSCs in the TME. 57 Autophagy could also induce the lysosomal breakdown of MHC-II and repress the antitumour effect of CD4 + T cells. 58 As a critical adaptive mechanism in a nutrient-poor environment, autophagy in the MDSCs and Tregs is essential to maintain their survival and sustained production of transforming growth factorβ (TGFβ), which dampens the activation of CTLs. 56,59,60 presentation to CD8 + T cells. 4,63,64 In addition, autophagy in transforming cells facilitates antigen presentation to CD8 + T cells. 65,66 Here, we summarize some evidence for the above notion and other potential mechanisms of autophagy that contribute to anti-tumour immunity.

| THE PROTEC TIVE ROLE OF AUTOPHAGY IN PROMOTING NEOANTI G EN PR E S E NTATI O N
The efficient uptake and presentation of tumour antigen is essential to subvert the immunosuppressant TME. Li and colleagues showed that tumour cell autophagy triggered by the synthetic Nano-DOX contributed to the increased immunogenicity of glioblastoma. These are presented as the elevated expression of MHC-I complex and antigen presentation on tumour cells, the activation of DCs, and the transmission of DAMPs into extracellular TME. 67 Michaud et al observed that autophagy of colon cancer cells could promote ICD, including the ATP release followed by IL1β released from activated DCs, and the latter cytokine might enhance DC functions. 53 Additionally, the autophagosome extruded by tumour cells, called TRAPs could also implicate in this process. One study indicated that TRAPs produced by alpha-tocopheryloxyacetic acid (a-TEA) treatment in breast and lung cancer models might boost the potential of DCs to intake and present antigens, then inducing the activation of CD8 + T cells. 65 Autophagy-mediated reduction of lysosomal integrity could potentiate MHC-I presentation and augment the cross-dressing of MHC-antigen complexes to DCs, contributing to significant CD8 + T-cell activation. 68 To address the tumour stagedependent dichotomous roles of autophagy, genetically engineered mouse models offer a robust tool. For example, in the early stage of carcinogenesis of KRas G12D murine lung cancer, autophagy inhibited Treg infiltration through suppressing adenosinergic signalling and repressed tumour growth. 61 However, the autophagy at later stage potentiated tumour progression via dampening oxidative stress as well as inhibiting the DNA damage response. 61 Similar to the observation in tumour cells, autophagy in macrophages was shown to promote the surface expression of MHC-II. 69 In a diethylnitrosamine-induced hepatocellular carcinoma model, autophagy in macrophages was essential for their intratumoral infiltration. 70 Another study reported that autophagy of T cells induced by metformin in a breast cancer model of mice could substantially enhance the functional CD8 + T-cell response by maintaining T-cell function; meanwhile, the autophagy of CD8 + memory T cells is considered indispensable to maintain their survival and sustain tumour immunosurveillance after tumour resection. 71

| THE CRUCIAL ROLE OF MITOPHAGY IN REG UL ATING TUMOUR IMMUNE RE S P ONS E
Autophagy-mediated turnover of aged and/or damaged mitochondria is known as mitophagy. 72,73 The role of mitophagy in modulating the tumour immunity is emerging. On one side, Ziegler and colleagues show that mitophagy promotes anti-tumour immunity.

Increased mitophagy in intestinal epithelial cells triggers iron
accumulation-induced lysosomal membrane permeabilization, which promotes the release of proteases into the cytosol and augments of MHC class I presentation. 68

| CON CLUS IONS
In summary, despite the dichotomous functions of autophagy in regulating anti-tumour immune responses, its predominant function is likely dependent on cancer stages, cancer types, immune infiltration profiles and modelling methods. Autophagy in immune cells is an essential protective mechanism by facilitating tumour neoantigen presentation. However, autophagy in cancer cells may promote adaptive resistance to immune killing by dampening IFN-I-mediated immune sensing and rapid turnover of cytotoxic effector molecules.
Global inhibition of autophagy may not yield the maximal benefits due to its interference with the antigen presentation machinery in the APCs; even such inhibition may sensitize tumours to immune killing. Thus, the characterization of specific genes regulating tumour immunogenicity and innovation in targeted delivery of autophagy inhibitors into tumour cells are among the most urgent tasks to sensitize cold cancers to immunotherapy.

ACK N OWLED G M ENTS
We would like to thank all grant supports, including the National Natural Science Foundation of China (81902782, 81520108009, 81621062, 81730030), the 111 MOE project of China (B14038), NIH R01 DE026728 and U01 DE029255.

CO N FLI C T O F I NTE R E S T
The authors declare no potential conflicts of interest.