Published August 30, 2019, doi:10.4049/jimmunol.1900475 The Journal of Immunology Frequent Loss of IRF2 in Cancers Leads to Immune Evasion through Decreased MHC Class I Antigen Presentation and Increased PD-L1 Expression Barry A. Kriegsman,* Pranitha Vangala,† Benjamin J. Chen,* Paul Meraner,‡ Abraham L. Brass,‡,x,{ Manuel Garber,† and Kenneth L. Rock* To arise and progress, cancers need to evade immune elimination. Consequently, progressing tumors are often MHC class I (MHC-I) low and express immune inhibitory molecules, such as PD-L1, which allows them to avoid the main antitumor host defense, CD8+ T cells. The molecular mechanisms that led to these alterations were incompletely understood. In this study, we identify loss of the transcription factor IRF2 as a frequent underlying mechanism that leads to a tumor immune evasion phenotype in both humans and mice. We identified IRF2 in a CRISPR-based forward genetic screen for genes that controlled MHC-I Ag presentation in HeLa cells. We then found that many primary human cancers, including lung, colon, breast, prostate, and others, frequently downregulated IRF2. Although IRF2 is generally known as a transcriptional repressor, we found that it was a transcriptional activator of many key components of the MHC-I pathway, including immunoproteasomes, TAP, and ERAP1, whose transcrip- tional control was previously poorly understood. Upon loss of IRF2, cytosol-to–endoplasmic reticulum peptide transport and N-terminal peptide trimming become rate limiting for Ag presentation. In addition, we found that IRF2 is a repressor of PD-L1. Thus, by downregulating a single nonessential gene, tumors become harder to see (reduced Ag presentation), more inhibitory (increased checkpoint inhibitor), and less susceptible to being killed by CD8+ T cells. Importantly, we found that the loss of Ag presentation caused by IRF2 downregulation could be reversed by IFN-stimulated induction of the transcription factor IRF1. The implication of these findings for tumor progression and immunotherapy are discussed. The Journal of Immunology, 2019, 203: 000–000. he importance of adaptive immunity in preventing cancer mutagen-induced tumors (1–3). In addition, tumors derived from was revealed through studies in which immunodeficient such immunodeficient animals grew when transplanted into other T animals, such as those lacking IFN-g, perforin, or RAG-2, immunodeficient hosts but were rejected when placed into im- werefoundtohaveamarkedincreaseinspontaneousand munocompetent hosts (3–5), providing further evidence that the immune system recognized such tumors and could reject them. In contrast, many tumors arising in immunocompetent animals *Department of Pathology, University of Massachusetts Medical School, Worcester, grew after being transplanted into immunocompetent hosts (3–5), MA 01655; †Department of Bioinformatics and Computational Biology, University of Massachusetts Medical School, Worcester, MA 01655; ‡Department of Microbi- thereby showing that cancers that arise and successfully progress ology and Physiological Systems, University of Massachusetts Medical School, in the face of the immune system have undergone immunoediting Worcester, MA 01655; xDepartment of Medicine, Gastroenterology Division, Uni- { to escape from immune control. This immunoediting process is versity of Massachusetts Medical School, Worcester, MA 01655; and Peak Gastro- enterology Associates, Colorado Springs, CO 80907 thought to be why many cancers express low levels of MHC class ORCIDs: 0000-0002-4146-2370 (B.A.K.); 0000-0003-4000-7891 (P.V.); 0000-0003- I (MHC-I) and upregulate certain inhibitory molecules (6). The 0153-4680 (P.M.). underlying molecular mechanisms responsible for these changes Received for publication April 29, 2019. Accepted for publication August 1, 2019. are poorly understood but have obvious potential impact on tumor This work was supported by National Institutes of Health Grants R01 AI114495 progression and immunotherapy (7, 8). (to K.L.R.), T32 AI095213 (to B.A.K.), and T32 GM107000 (to B.A.K.). This work The MHC-I presentation pathway is critical for immune rec- was funded by an Investigators in the Pathogenesis of Infectious Disease grant from ognition and elimination of tumors by CD8+ T cells. In this the Burroughs Wellcome Fund (to A.L.B.). A.L.B. is grateful to the Bill and Melinda Gates Foundation and to Gilead Sciences Inc. for their support. process, a fraction of peptides that are generated by proteaso- The RNA-sequencing data presented in this article have been deposited to the Gene mal degradation of cellular proteins are transported by the TAP Expression Omnibus under accession number GSE133089 (www.ncbi.nlm.nih.gov/ transporter into the endoplasmic reticulum (ER), wherein they can geo/query/acc.cgi?acc=GSE133089). be further trimmed by the aminopeptidase, ERAP1 (9–11). Sub- Address correspondence and reprint requests to Dr. Kenneth L. Rock, University of sequently, peptides of the correct length and sequence bind to Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655. E-mail address: [email protected] MHC-I molecules and these complexes are then transported to the + The online version of this article contains supplemental material. cell surface for display to CD8 T cells. This allows activated CD8+ T cells to identify and kill cells that are presenting tumor- Abbreviations used in this article: ChIP, chromatin immunoprecipitation; Ct, cycle threshold; DC, dendritic cell; ER, endoplasmic reticulum; EV, empty vector; IP, specific peptides (e.g., from mutant proteins) on their MHC-I (12). immunoprecipitation; IRF2-KO, IRF2-knockout; ISRE, IFN-stimulated response We performed an unbiased, forward genetic screen in human element; MFI, mean fluorescence intensity; MHC-I, MHC class I; MHC-II, MHC class II; NEB, New England Biolabs; NSCLC, non–small cell lung cancer; PD-L1, cervical carcinoma HeLa H1 cells to identify genes whose loss programmed death-ligand 1; qPCR, quantitative PCR; RNA-seq, RNA sequencing; downregulated the MHC-I pathway. In this screen, the second RT, room temperature; sgRNA, short guide RNA; siRNA, small interfering RNA; strongest hit, second only to b2-microglobulin (the MHC-I WT, wild-type. L chain), was IRF2, an IFN regulatory transcription factor Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 that had not been previously recognized to positively regulate this www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900475 2 FREQUENT LOSS OF IRF2 IN CANCERS LEADS TO IMMUNE EVASION pathway. In this article, we show that under basal conditions, 59-GACTACCGGTATGCCGGTGGAAAGGATGCGCATG-39;humanIRF2 not only does IRF2 positively regulate the MHC-I pathway by sgRNA mut reverse: 59-GCCGTGCCTCGCTGCGTGCATCCAGGGGATCT- transcriptionally activating genes necessary for peptide transport GAAAAATCTTCTTTTCCTTG-39; human IRF2 sgRNA mut forward: 59-GATGCACGCAGCGAGGCACGGCTGGGATGTGGAAAAAGATGCA- and processing, but it also transcriptionally represses the ex- CCACTCTTTAGAAA-39; human IRF2 MluI reverse: 59-GATCACGCGTTTA- pression of programmed death-ligand1(PD-L1),animportant ACAGCTCTTGACGCGGGCCTGG-39; mouse IRF2 AgeI forward: immune checkpoint molecule. These results, together with our 59-GATCACCGGTATGCCGGTGGAACGGATGCGAATG-39;mouse findings that many human cancers have downregulated IRF2 IRF2 sgRNA mut reverse: 59-CCTTTTTTCGAGGGGCGCTCTGATAA- GGGCAGCATCCGGTAGACTCTGAAGGCG-39; mouse IRF2 sgRNA expression and that tumor cells lacking IRF2 are more difficult to mut forward: 59- CTTATCAGAGCGCCCCTCGAAAAAAGGAAAGA- kill, demonstrate that loss of IRF2 by cancers is a common immune AACCAAAGACAGAAAAAGAAGAGAG-39; and mouse IRF2 MluI re- evasion mechanism and this has obvious therapeutic implications. verse: 59-GATCACGCGTTTAACAGCTCTTGACACGGGCCTGG-39. Cell surface staining Materials and Methods Cells Where indicated, mouse cells were blocked with 2.4G2 and stained for surface MHC-I levels with anti-Kb–allophycocyanin (AF6-88.5.5.3; A E DC3.2 is a J2 virus-immortalized dendritic cell (DC) line (13). A particular eBioscience), MHC-II levels with anti–I /I -PECy7 (M5/114.15.2; Bio- DC3.2 clone (with Renilla luciferase) was used for all experiments in this Legend), PD-L1 levels with anti–PD-L1-PE (10F.9G2; BioLegend), or study, as this clone has very strong cross-presentation and MHC class II with isotype controls (eBioscience mouse IgG2a-allophycocyanin eBM2a, (MHC-II) presentation, as compared with other clones. RF33.70 is a T cell eBioscience rat IgG2b k-PE eB149) at 1:200 dilutions. Where indicated, hybridoma that recognizes the OVA peptide OVA257–264 in the context of human cells were stained for surface MHC-I levels with W6/32. W6/32 b H2-K (14). MF2.2D9 is a T cell hybridoma that recognizes OVA258–276 in staining was performed either by two-step labeling with W6/32 hybridoma the context of I-Ab (13). RF33.70 and MF2.2D9 were transduced with supernatant followed by 1:500 donkey–anti-mouse Alexa 647 (Life lentivirus containing NFAT-luciferase. NIH-3T3 cells were stably trans- Technologies) or by one-step labeling with 1:200 FITC-conjugated W6/32 fected with the mouse H2-Kb molecule. A549 and MCF7 were kindly (eBioscience). Where indicated, human cells were stained for surface provided by Leslie Shaw (University of Massachusetts), and the D53m and PD-L1 levels with 1:200 rabbit anti–PD-L1 (28-8; Abcam), followed by H50m mouse MCA-induced sarcoma lines were kindly provided by 1:500 donkey–anti-rabbit Alexa 647 (Life Technologies). Normalized R. Schreiber (Washington