HPV Epitope Processing Differences Correlate with ERAP1 Allotype and Extent of CD8 T-Cell Tumor Infiltration in OPSCC

Published OnlineFirst May 31, 2019; DOI: 10.1158/2326-6066.CIR-18-0498

Research Article Cancer Immunology Research HPV Epitope Processing Differences Correlate with ERAP1 Allotype and Extent of CD8þ T-cell Tumor Inﬁltration in OPSCC Emma Reeves1,2, Oliver Wood1,2, Christian H. Ottensmeier1,2, Emma V. King1,2, Gareth J. Thomas1,2, Tim Elliott1,2, and Edward James1,2

Abstract

Presence of tumor-infiltrating lymphocytes (TIL) predicts allotype combinations identified in OPSCC correlate with þ survival in many cancer types. In HPV-driven cancers, cervical tumor-infiltrating CD8 T-cell (CD8)/TIL (CD8/TIL) status and oropharyngeal squamous cell carcinomas (CSCC and of the tumor. Functional analyses revealed that ERAP1 OPSCC, respectively), numbers of infiltrating T cells, particu- allotype combinations associated with CD8/TILlow tumors þ larly CD8 T cells, and presentation of HPV E6/E7 epitopes are have a reduced capacity to generate both a model antigen associated with improved prognosis. Endoplasmic reticulum SIINFEHL and the HPV-16 E782-90 epitope LLMGTLGIV from aminopeptidase 1 (ERAP1) regulates the presented peptide N-terminally extended precursor peptides. In contrast, ERAP1 repertoire, trimming peptide precursors prior to MHC I load- allotypes from CD8/TILhigh tumors generated the epitopes ing. ERAP1 is polymorphic, and allotypic variation of ERAP1 efficiently. These data reveal that ERAP1 function correlates enzyme activity has an impact on the presented peptide with CD8/TIL numbers and, by implication, prognosis, sug- repertoire. Individual SNPs are associated with incidence and gesting that the presentation of HPV-16 epitopes at the cell outcome in a number of diseases, including CSCC. Here, we surface, resulting in an anti-HPV T-cell response, may depend highlight the requirement for ERAP1 in the generation of HPV on the ERAP1 allotype combinations expressed within an E6/E7 epitopes and show that the functional activity of ERAP1 individual.

expressed by the virus (5, 7, 8). Furthermore, more tumor- Introduction þ þ infiltrating CD8 T cells (tumor-infiltrating lymphocytes, TIL) in Lymphocyte infiltration of tumors, in particular by CD8 T OPSCC are associated with improved survival (5, 9). cells, is associated with better clinical outcome in many þ The presentation of peptide epitopes to CD8 T cells is depen- cancers (1–6). Although this tumor infiltration is driven by a dent on the antigen-processing pathway. Several components of number of factors such as expression of chemokines and their this pathway are polymorphic, including HLA, endoplasmic receptors, the presentation of immunogenic peptide epitopes is reticulum aminopeptidase 1 (ERAP1), the transporter associated paramount. The identity of immunogenic peptide epitopes and þ with antigen processing (TAP), and to a lesser extent Tapasin (10). hence, specificity of tumor-infiltrating CD8 T cells, has not been ERAP1 trims N-terminally extended peptide epitopes to the determined in most instances, but it is likely that T-cell specificity correct length for optimal binding to HLA and therefore serves and efficacy are linked, as they are in other diseases. However, in as an editor of the peptide repertoire presented at the cell surface. human papillomavirus (HPV)-driven cancers such as cervical We have previously shown that ERAP1 allotypes arising from its squamous cell carcinoma (CSCC) and oropharyngeal squamous polymorphism can differ in their peptide trimming function, cell carcinoma (OPSCC), where HPV infection accounts for >95% defined by both overall activity and specificity for the N-terminal and 40% to 80% of cases in the western world respectively, þ amino acid of peptide epitope precursors (11). In CSCC, genome- peripheral and tumor-infiltrating CD8 T cells have been shown wide association studies (GWAS) showed nonsynonymous exon- to recognize epitopes derived from the E6/E7 oncoproteins ic SNPs of ERAP1 were associated with disease incidence (12), with 2 of these SNPs associated with disease-free and overall survival (13). Given that CSCC is predominantly driven by HPV 1Centre for Cancer Immunology, University of Southampton Faculty of Medicine, 2 infection and, similar to OPSCC, shows improved survival with University Hospital Southampton, Southampton, United Kingdom. Institute for þ more TIL (14), the ability to control HPV tumors is likely Life Sciences, University of Southampton, Southampton, United Kingdom. þ mediated by CD8 T cells specific for HPV-derived antigens (4, 5). Note: Supplementary data for this article are available at Cancer Immunology Thus, the ability to generate and present peptide epitopes derived Research Online (http://cancerimmunolres.aacrjournals.org/). from viral proteins, such as E6 and E7, likely drives antitumor Corresponding Author: Edward James, Centre for Cancer Immunology, responses. University Hospital Southampton, MP127, Tremona Road, Southampton SO16 In this study, having observed similarities in SNP variants 6YD, United Kingdom. Phone: 4423-8021-5884; Fax: 4423-8120-5152; E-mail: þ [email protected] between CSCC and a cohort of HPV patients with OPSCC, we tested the hypothesis that allotypic variation in ERAP1 might Cancer Immunol Res 2019;7:1202–13 þ underpin the extent of CD8 T-cell tumor infiltration via its doi: 10.1158/2326-6066.CIR-18-0498 impact on processing of tumor-specific (HPV-derived) epitopes. 2019 American Association for Cancer Research. In individual patients, we found that ERAP1 function correlates

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þ with tumor infiltrating CD8 T cell (CD8)/TIL (CD8/TIL) status in CATTAACAGSNNCGCGCTGCAGACTGCCGC-30, where N ¼ any resected tumors: ERAP1 in CD8/TILlow tumors had reduced nucleotide and S ¼ C or G. Constructs were sequence verified, and capacity to generate the HPV E7-derived epitope, LLMGTLGIV, the most frequent codon for each amino chosen for use where þ suggesting that the lack of CD8 T-cell infiltration may be linked possible. to the ability to generate and present virally derived peptide epitopes. These data suggest that ERAP1 function may identify Dextramer detection of HPV-specific T cells "high-risk" patients with OPSCC with poor prognosis. Patient tumor samples were mechanically disaggregated into single-cell suspensions before being analyzed for the presence of Materials and Methods HPV16-specific T cells by flow cytometry. Dextramers specific for HPV16 E6 (KLPQLCTEL; KL9), E6 (TIHDIILEV; TV10), fi 18-26 29-38 Patients, HPV classi cation, and CD8/TIL scoring and E7 (LLMGTLGIV; LV9) were obtained from Immudex fi þ 82-90 Patients previously classi ed for HPV tumor status using HPV and directly conjugated with PE, APC, and FITC fluorophores, ISH and p16 immunochemistry were used in this study (REC respectively. Tumor cells were preincubated with 50 nmol/L reference 09/H0501/90). Tumor histology was previously char- protein kinase inhibitor dasatanib for 30 minutes at 37C, before acterized for each patient (9) and reviewed for this study by the addition of 10 mL dextramer and incubated for a further 30 pathologist GJT according to the 1998 UK Royal College of minutes at 4C. After two washes, surface antibodies for CD8 fi Pathologist Guidelines. Lymphocyte in ltrate (TIL) was deter- (anti-CD8-Pacific Blue, clone RPA-T8; BD Biosciences) and CD3 fi mined and scored under low-power magni cation ( 2.5 objec- (anti-CD3-AmCyan, clone SK7; BD Biosciences) were incubated tive) as high (diffuse; present in >80% of tumor/stroma), mod- for 20 minutes at 4C before washing. Cells were acquired on BD – erate (patchy; present in 20% 80% of tumor/stroma), or low FACS Canto II (BD Biosciences) and analyzed using Flow Jo (weak/absent; present in <20% of tumor/stroma; ref. 9). In þ Software (TreeStar Inc.). addition, CD8 T-cell infiltration was scored by IHC and enu- þ meration of CD8 T cells was expressed as an average of 10 high- power fields (9). Images were collected using a CKX41 inverted Generation of BE7A2Z hybridoma microscope with DP-22 color camera running under CellSens The generation of LacZ-inducible T-cell hybridomas has been described previously (15). Briefly, the BE7A2Z hybridoma was (Version2) software and 10 or 40 achromat objective lenses (all Olympus; Supplementary Fig. S1A). Grouping was defined by generated by immunizing HLA-A 0201 transgenic HHD mice þ the tertile values: high (>39 CD8 T cells/field), moderate (>13 with LLMGTLGIV-pulsed BMDC and splenocytes fused with the þ þ and <39 CD8 T cells/field), and low (<13 CD8 T cells/field; BWZ.36/CD8a cell line as described previously (15). To deter- fi Supplementary Fig. S1B). mine peptide speci city of the hybridoma, splenocytes were pulsed with 10 mmol/L LV9 or 10 mmol/L irrelevant peptide ERAP1 isolation and identification and hybridomas assessed for IFNg production. To determine fi ERAP1 was amplified from whole blood samples and cloned sensitivity of subsequent LV9-speci c clones, 293T cells expres- into the pcDNA3.1 expression vector (Life Technologies) as sing endogenous HLA-A2 were pulsed with 10 nmol/L LV9 described previously (11). ERAP1 clones from each patient were peptide and cocultured with hybridoma clones for 24 hours. Each sequenced (Source Bioscience Ltd.) and compared with the wild- hybridoma clone sensitivity to LV9 was assessed by type ERAP1 sequence (UniProt – Q9NZ08) to identify the pres- measurement of intracellular LacZ activity by chlorophenol ence of polymorphic variants using DNADynamo alignment red-b-d-galactopyranoside (CPRG; Roche). software (Blue Tractor Software). To further validate the presence of ERAP1 polymorphisms, gDNA was extracted from whole blood Cell lines, transfection, and T-cell activation assay using DNeasy Blood and Tissue Kit (Qiagen) and targeted ERAP1 Culture conditions for ERAP1 knockout HEK293T (293T exons amplified and sequenced using the following primers: E1KO) and T-cell hybridoma cells have been described M349V 5-GATGATTGTTTGGGAGAATG-30 and 50-GCCTTA- before (16). 293T E1KO cells were cultured from freshly thawed TATGTCCACGCT-30; K528R 50-CTTTCAGTCTGGTGCTATGG-30 aliquots from passage 2 (generated from original 293T cell line in and 50-GTGTGATGGCTGGGGACATC-30; D575N 50-GCTCTACT- 2013) and used over 2 weeks for the indicated experiments. Cells CAAGGAGTCCAAG-30 and 50-CGATTTTCAATCTGATCCC-30; were authenticated by STR analysis and were confirmed Myco- and R725Q/Q730E 50-CCTGACTTTAAGTAGATGG-30 and 50- plasma free. For assessment of ERAP1 trimming activity, 293T GGTTATTTAGTAAGGACTGAC-30. E1KO cells were transfected with DNA using FuGENE 6 Trans- fection Reagent (Promega). For assessment of single amino acid DNA constructs trimming specificity, 293T E1KO cells were transfected with 0.1 mg The pcDNA3.1 minigenes (ES)-SHL8, (ES)-X-SHL8, H2-Kb, total DNA, consisting of 0.05 mg E320A nonfunctional ERAP1 or ERAP1002 as well as the nonfunctional active site ERAP1 0.025 mg of each patient ERAP1 allotype, together with 0.025 mg mutant, E320A, have been described previously (11). The HPV- H2-Kb (SHL8) or HLA-A0201 (LV9) and 0.025 mg X-SHL8 or 16 E7 epitope-specific construct (ES)-LV9 was generated by the X-LV9. For assessment of LV9 extended precursor trimming (ED/ insertion of oligonucleotides into the EcoRI/XbaI sites of D-LV9), 293T E1KO cells were transfected with 1 mg total DNA, pcDNA3.1, encoding the ER translocation signal sequence (ES) consisting of 0.5 mg E320A or 0.25 mg each patient ERAP1 allotype, followed by the amino acid sequence LLMGTLGIV (LV9). The together with 0.25 mg ED/D-LV9 or LV9 and 0.25 mg HLA-A0201. (ES)-X-LV9 constructs were generated by site directed mutagenesis All transfected cells were incubated for 24 hours at 37C. Presen- to incorporate a single amino acid into the (ES)-LV9 construct, tation of final peptide SHL8 or LV9 and activation of the respective using the following primers: 50-GCGGCAGTCTGCAGCG- LacZ-inducible T-cell hybridoma, B3Z or BE7A2Z, was assessed by CGNNSCTGTTAATGGGCACACTAG-30 and 50- CTAGTGTGCC- measurement of intracellular LacZ activity by CPRG.

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Statistical analysis Table 1. ERAP1 allotype identity in patients with OPSCC Two or one-way ANOVA with Dunnett posttest was performed Amino acid at indicated position for analysis of differences between multiple groups and controls Patient CD8/TIL ERAP1 56 127 346 349 528 575 725 730 number status allotype E R G M K D R Q (GraphPad Prism). 1 High 001 E P G VRNQE 002 E R G M K D R Q 2 High 011 E R G M R DRE Results 016 E R G V KDQE ERAP1 allotype sequences in patients with OPSCC 3 High 001 E P G VRNQE ERAP1 018 E R G M K D R E polymorphism is associated with disease risk and disease-free survival in HPV-induced CSCC (12, 13). For OPSCC, 4 High 001 E P G VRNQE 002 E R G M K D R Q 40% to 80% of cases in the western world are also associated with 5 High 001 E P G VRNQE HPV infection and within this group of patients, the amount of 002 E R G M K D R Q tumor-infiltrating lymphocytes (TIL) observed in resected tumors 6 High 002 E R G M K D R Q correlates with disease survival (9, 17). We therefore investigated 002 E R G M K D R Q ERAP1 7 High 002 E R G M K D R Q whether patients with OPSCC shared the same polymorphisms observed in CSCC and whether the allotypes 014 KP GMR DRE 8 High 001 E P G VRNQE expressed in patients correlated with numbers of tumor- þ 011 E R G M R DRE fi ERAP1 in ltrating CD8 T cell (CD8)/TIL. We sequenced both 9 High 011 E R G M R DRE copies expressed in a cohort of 26 patients with OPSCC whose 015 E P GMR DRE tumors had been scored with high, moderate, or low numbers of 10 High 001 E P G VRNQE CD8/TIL. Two of the CSCC-associated ERAP1 SNPs (P127 and 002 E R G M K D R Q 11 Moderate 015 E P GMR DRE E730) were detected in the majority of patients, regardless of CD8/ ¼ 015 E P GMR DRE TIL status, with E730 being more frequently observed (E730 9 12 Moderate 001 E P G VRNQE high mod low of 10 CD8/TIL , 6 of 7 CD8/TIL and 7 of 8 CD8/TIL 011 E R G M R DRE high mod patients; P127 ¼ 8 of 10 CD8/TIL , 2 of 7 CD8/TIL , and 4 of 13 Moderate 002 E R G M K D R Q 8 CD8/TILlow patients; Table 1). The number of patients that 017 E R G M K N RQ contained both P127 and E730 varied between CD8/TIL groups 14 Moderate 011 E R G M R DRE high 018 E R G M K D R E with CD8/TIL having the greatest proportion (8/10 vs. 2/7 mod low 15 Moderate 002 E R G M K D R Q CD8/TIL and 4/8 CD8/TIL ; Table 1). Examination of the 2 011 E R G M R DRE individual ERAP1 allotypes from each patient that contained 16 Moderate 011 E R G M R DRE either of the P127 or E730 variants in a single molecule (CD8/ 011 E R G M R DRE TILhigh ¼ 13/20 total allotypes; CD8/TILmod ¼ 11/14; CD8/TILlow 17 Moderate 016 E R G V KDQE ¼ 12/16), revealed that a proportion did not contain both of the 018 E R G M K D R E mod 18 Low 007 E R G M R D Q Q variants (P127 and E730) in the same molecule, with CD8/TIL low high 011 E R G M R DRE and CD8/TIL having the lowest frequency (8/13 TIL , 3/11 19 Low 001 E P G VRNQE mod low TIL , 4/12 TIL ; Table 1). 019 E R D M R DRE Overall, most of the allotypes observed have been previously 20 Low 001 E P G VRNQE described (001; Hap10, 002; Hap2, 007, 011; Hap4, 013; 001 E P G VRNQE Hap1, 014; Hap7, 015; Hap6, 018; Hap3 and 019; 21 Low 011 E R G M R DRE 011 E R G M R DRE Hap5; Table 1; refs. 16, 18). Two allotypes that have not previ- fi 22 Low 013 E P GMKDRQ ously been identi ed ( 016, 017). Comparison of allotypes from 019 E R D M R DRE different CD8/TIL groups showed that 5 allotypes were only 23 Low 002 E R G M K D R Q found in one of the 3 groups; however, almost all of these 002 E R G M K D R Q represented a single incidence (Supplementary Table S1). Three 24 Low 002 E R G M K D R Q 011 E R G M R DRE allotypes were observed in all CD8/TIL groups and three in two groups (Supplementary Table S1), showing that, for this cohort, 25 Low 011 E R G M R DRE 011 E P GMR DRE analysis of individual ERAP1 allotypes did not correlate with NOTE: Bold type indicates variants at the indicated amino acid position. CD8/TIL status.

CD8/TILlow allotype combinations have poor trimming function that stimulates T cells to the same degree as the original peptide We have previously shown, for an autoinflammatory disease, (Supplementary Fig. S2), from precursor peptides with single that the strongest association between ERAP1 and disease is amino acid N-terminal extensions in living cells. The SHL8/Kb achieved by considering ERAP1 allotype pairing, because this complexes are detected at the cell surface by the specific T-cell best represents the combined enzyme function of the codomi- hybridoma, B3Z. Briefly, ERAP1 allotypes fall into three general nantly expressed allotypes (16). We therefore investigated the classes of activity: those that process the epitope efficiently, those peptide-editing function of ERAP1 allotype combinations in that are hypofunctional, and those that are hyperfunctional and patients with OPSCC, utilizing the model system previously used overtrim the epitope, thus destroying it. Further differentiation of to assess trimming activity (11). This system classified ERAP1 function is seen in N-terminal specificity. This assay is preferable allotypes with respect to their differential ability to generate a to in vitro assays of function using small chromogenic substrates model epitope, SIINFEHL (SHL8) a modified form of SIINFEKL because it evaluates function in vivo, in the context of a functioning

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antigen-processing pathway, and provides a readout that relates and moderate infiltrated tumors (Fig. 1D), high and low infil- directly to the impact on antigen presentation. trated tumors (Fig. 1E), and in both moderate and low infiltrated We analyzed the enzyme activity of ERAP1 allotype combina- tumors (Fig. 1F). The activity toward each substrate was expressed tions that were observed exclusively in either high, moderate, or as a percentage of the maximal activity observed with the SHL8 low infiltrated tumors using this assay (Fig. 1A–C). Each allotype minigene (which does not require processing by ERAP1). By tested gave rise to a characteristic activity signature according to calculating the total activity across all substrates for each allotype their relative ability to cleave X from the precursor substrate combination as AUC, we found that allotype combinations that (where X is any one of the 20 naturally occurring amino acids). were associated exclusively with low infiltrated tumors had a We also measured the activity of allotypes observed in both high significantly reduced activity compared with those associated with

A CD8/TIL high *001 + *002 100 100 100 *001 + *018 Patient: #1, #4, #5, #10 *002 + *014 Patient: #3 80 80 Patient: #7 80

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0 0 0 ACDEFGHI KLMNPQRSTVWY ACDEFGHI KLMNPQRSTVWY ACDEFGHI KLMNPQRSTVWY B CD8/TIL moderate 100 *015 + *015 100 *002 + *017 100 *011 + *018 Patient: #11 Patient: #13 Patient: #14 80 80 80

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0 0 0 ACDEFGHI KLMNPQRSTVWY ACDEFGHI KLMNPQRSTVWY ACDEFGHI KLMNPQRSTVWY C CD8/TIL low 100 *007 + *011 100 *001 + *019 100 *001 + *001 80 Patient: #18 80 Patient: #19 80 Patient: #20

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0 0 0 ACDEFGHI KLMNPQRSTVWY ACDEFGHI KLMNPQRSTVWY ACDEFGHI KLMNPQRSTVWY D CD8/TIL high and moderate E CD8/TIL high and low F CD8/TIL moderate and low *001 + *011 *002 + *002 *002 + *011 100 Patient: #8, #12 100 Patient: #6, #23 100 Patient: #15, #24 80 80 80

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0 0 0 ACDEFGHI KLMNPQRSTVWY ACDEFGHI KLMNPQRSTVWY ACDEFGHI KLMNPQRSTVWY Amino acid (X-SHL8)

Figure 1. N-terminal amino acid trimming speciﬁcity by OPSCC ERAP1 allotype combinations. E1KO 293T cells were transfected with ERAP1 allotype combinations from patients with OPSCC together with H2-Kb and X-SHL8 minigenes representing 20 amino acids and assessed for generation of SHL8 by B3Z activation. OPSCC ERAP1 allotype identity from patients with TIL status are shown in (A) CD8/TILhigh,(B) CD8/TILmoderate,(C) CD8/TILLow,(D) CD8/TILhigh and CD8/TILmoderate,(E) CD8/TILhigh and CD8/TILlow, and (F) CD8/TILmoderate and CD8/TILlow. The relative presentation of trimmed X-SHL8 was compared with that of the maximal response using SHL8, which does not require ERAP1 activity. Data pooled from 4 independent experimental repeats SEM.

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high or moderate infiltrated tumors (50% and 55%, uncharged polar). The results in Fig. 2C show that reduced respectively; Fig. 2A and B). activity of allotype combinations seen in CD8/TILlow tumors is We next analyzed the specificity of each ERAP1 allotype com- observed across all classes of N-terminal amino acid, closer bination by considering their activity against N-terminal exten- inspection suggests that precursors with charged, polar sions classified according to their physicochemical properties uncharged, or aliphatic hydrophobic N-1 amino acids are most (aliphatic hydrophobic, aromatic hydrophobic, basic, acidic, effected (charged ¼ 3/5 residues, polar uncharged ¼ 3/4 residues,

A B **** **** Allotype CD8/TIL Overall SHL8 **** Mean AUC 1,200 ** * combination status generation (AUC) * *002+*014 High 812.1 *001+*002 “ 954.8 813 900 **** *001+*018 “ 672.1 *001+*011 High + Mod 581.9 581.9 *002+*002 High + Low 930.3 930.3 600 *002+*011 Mod + Low 1039.1 1039.1 *002+*017 Mod 714.3 Mean AUC 300 *011+*018 “ 892.5 748.8 *015+*015 “ 639.6 *007+*011 Low 415.3 0 *001+*019 “ 338.1 410.0 h d d *001+*001 “ 476.5 o w w o ig /lo lo Low H m h d/ M h/ o ig M H Hig CD8/TIL status

C Basic Acidic Polar uncharged H N 100 *** 80 80 ** *** K D * Q R S 80 ** * E ** * 60 * 60 * T 60 40 40 40 20 20 20

CD8/ 0 0 0 High Low Mod High Low Mod High Low Mod TIL status Hydrophobic Aliphatic Aromatic Special A 80 * 40 F 60 C ** I W G % Maximal response * * ** L Y 60 V 30 40

40 20

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CD8/ 0 0 0 High Low Mod High Low Mod High Low Mod TIL status

Figure 2. ERAP1 allotype combinations from CD8/TILlow tumors have reduced trimming activity. A, The total trimming activity for all 20 X-SHL8 substrates for each allotype combination were combined and grouped into CD8/TIL status. Overall trimming for each CD8/TIL group is represented as AUC. B, Table showing both the individual AUC for allotype pairs in each CD8/TIL group and the mean for each group. C, Speciﬁcity of ERAP1 trimming activity from those found exclusively in CD8/TILhigh, CD8/TILmoderate, or CD8/TILlow tumors towards X-SHL8 amino acids grouped based on physicochemical properties; basic, acidic, polar uncharged, aliphatic, aromatic, and special. Data from A–C, pooled from 4 independent experimental repeats SEM (, P 0.001; , P 0.01; , P 0.05).

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aliphatic ¼ 2/4 residues were significantly reduced in CD8/TILlow Cell surface presentation of LV9 epitope is ERAP1 dependent vs. CD8/TILhigh). To investigate the effect of ERAP1 on the generation of LV9, we created a T-cell hybridoma recognizing LV9 restricted by HLA-A0201 by immunizing HLA-A0201 transgenic (HHD) þ Anti-HPV CD8 T-cell responses in OPSCC tumors mice with peptide-pulsed BMDC and fusing splenocytes har- þ These observations are consistent with the hypothesis that the vested at day 7 after the final immunization with BWZ.36 CD8a generation of epitopes requiring N-terminal trimming recruits cells (Supplementary Fig. S3A; ref. 15). Several hybridomas were þ CD8 T cells into the tumor mass and therefore that ERAP1 generated and screened on LV9-pulsed splenocytes (Supplemen- function is a determinant in this process. The data also suggest tary Fig. S3B). Four hybridomas were found to be peptide-specific þ that the impact of ERAP1 polymorphism on CD8 T-cell recruit- and were stimulated by peptide-pulsed HEK293T cells at the ment might be greatest for T cells recognizing epitopes generated nanomolar quantities, with hybridoma #4 (called BE7A2Z) from precursors with charged/polar uncharged/aliphatic hydro- showing the greatest sensitivity and used in subsequent experi- þ phobic amino acids. Better prognosis for HPV patients with ments (Supplementary Fig. S3C). To assess the in vivo processing OPSCC and CSCC is associated with more TIL (5, 9, 14) and there of the LV9 peptide, we transfected ERAP1 knockout HEK293T þ is evidence that CD8 T cells recognizing epitopes derived from (E1KO 293T) cells with a minigene encoding the final (LV9) the HPV E6/E7 proteins are among TILs in these cancers (5, 8, 19). peptide. This construct sensitized both ERAP1-sufficient and We therefore scanned the immediate upstream sequence of pre- E1KO 293T cells, whereas constructs encoding N-terminally dicted proteasomal cleavage products of E6 and E7 that contained extended LV9 (ED-LV9 and D-LV9) were generated only by a putative HLA-A 0201–restricted epitope (Table 2; refs. 20–22). ERAP1-sufficient cells, showing that in vivo, the processing of LV9 Of the 4 E6 and 4 E7-derived peptides that have been confirmed as from these 2 precursor peptides is ERAP1 dependent (Supple- epitopes in previous studies (7, 23), 3 consisted of a core sequence mentary Fig. S3D). with a predicted IC50 for HLA-A 0201 of less than 100 nmol/L. In addition, 5 of the 8 peptides had a predicted 1 or 2 amino acid CD8/TILlow allotype combinations have reduced functionality extension following proteasomal cleavage. However, only 1 of the and different peptide specificity epitopes had both an IC50 of less than 100 nmol/L and contained We next measured the activity of ERAP1 allotype pairs that we an N-terminal extension, HPV E7 82-90, LLMGTLGIV identified exclusively in CD8/TIL high, moderate, or low groups (LV9; Table 2). The N-terminal extension of LV9 is comprised of using X-LV9 minigene encoded substrates, as described above for acidic amino acids (Glu, Asp; ED-LV9) both of which were X-SHL8. Examination of the allotype pairs showed a similar low trimmed less efficiently by CD8/TIL allotype pairs when ana- distribution of activities as for X-SHL8, with allotype pairs from lyzed with the X-SHL8 substrate (Fig. 2C). In addition, the CD8/TILhigh and CD8/TILmod patients having a significantly orthologue of this sequence from HPV-type 11 has been con- higher overall activity than those from CD8/TILlow when the AUC firmed as an epitope in infected humans and so we selected LV9 was calculated (CD8/TILhigh ¼ 45% and CD8/TILmod ¼ 35% for further analysis (24). greater; Fig. 4A and B). Original data for the 9 allotype combina- The precise immunodominance hierarchy of HLA-A 0201– tions from CD8/TILhigh, CD8/TILmod, and CD8/TILlow groups are þ restricted anti-HPV CD8 T-cell responses has not been well shown in Supplementary Fig. S4A–S4C, respectively. Ala-, Cys-, þ characterized. To further confirm the role of LV9-CD8 T cells Met-, Thr-, and Tyr-LV9 trimming by ERAP1 had a high back- in antitumor immunity, we assessed the presence of anti-HPV ground for E320A ERAP1, making it difficult to confirm the þ responses in 5 HPV OPSCC tumors. Investigation of 3 E6/E7 amount of trimming of each allotype pair, consequently these peptide-specific responses, LV9, TV10, and KL9, revealed that data are not included. When we analyzed the specificity of responses to all peptides were observed (Fig. 3A and B), with N-terminus preference, we found significantly reduced responses those directed to LV9 and TV10 the most prevalent. Indeed, LV9- for CD8/TILlow compared with the other two CD8/TIL groups and TV10-specific responses were dominant in 2 tumors (#1 and among the charged, polar uncharged, and special N-terminal #4 vs. #3 and #5, respectively), and codominant in 1 tumor (#2) amino acid extensions (Fig. 4C). The trimming of Lys, Asp, and (Fig. 3B). These responses confirmed LV9 as a good candidate Glu was more efficient in the context of the LV9 peptide backbone peptide to investigate the role of ERAP1 in generation of HPV- compared with SHL8 for all CD8/TIL groups (Lys ¼ 15%–50% for derived antigenic peptides. SHL8, 60%–90% for LV9, Asp ¼ 15%–30% for SHL8, 50%–70% for LV9 and Glu ¼ 30%–50% for SHL8, 85%–90% for LV9; Figs. 2

and 4). Conversely, the aliphatic hydrophobic amino acids Ile and Table 2. HLA-A 0201–restricted HPV-16 E6/E7 peptide epitopes Leu were trimmed less efficiently in the context of LV9 than that MHC IC50 Predicted proteasome ¼ – – Position (predicted cleaved peptide observed for SHL8 (Ile 15% 40% SHL8, 5% 10% LV9, and (amino Epitope nmol/L) (N-terminal extension) Leu ¼ 30%–60% SHL8, 10%–20% LV9; Figs. 2 and 4). These Protein acid) sequence NetMHC NetCHOP, MAPPP results indicate that although there are differences in amino acid E6 7–15 AMFQDPQER — RTAMFQDPQER specificity between substrates X-SHL8 and X-LV9, the total activity E6 18–26 KLPQLCTEL 134.4 PRKLPQLCTEL of CD8/TILlow ERAP1 allotype pairs is reduced compared with – E6 29 38 TIHDIILECV 200.6 QTTIHDIILECV/ that from CD8/TILhigh patients. TIHDIILECV E6 42–50 FAFRDLCIV 100.4 DFAFRDLCIV low E7 7–15 TLHEYMLDL 94.1 TLHEYMLDL Generation of LV9 peptide epitope is impaired in CD8/TIL E7 11–19 (20) YMLDLQPET(T) 10.58 (33.3) YMLDLQPET(T) ERAP1 allotype pairs þ E7 82–90 LLMGTLGIV 37.11 EDLLMGTLGIV/ The best prognosis for HPV OPSCC is associated with high þ DLLMGTLGIV infiltration of CD8 T cells into the tumor (9), many of which are – E7 86 93 TLGIVCPI 409.85 TLGIVCPI HPV specific (Fig. 3; refs. 5, 19, 25). With the reduced trimming

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A – Dextramer + Dextramer

B LV9 4 LV9 3 TV10 Figure 3. Presence of anti-HPV E6/E7 epitope CD8þ T-cell KL9 responses in HPVþ OPSCC tumors. Tumors from HPVþ 2 patients with OPSCC were assessed for the presence TV10 þ

dextramer+ cells of CD8 T-cell responses to the HLA-A 0201-

+ restricted HPV E6/E7 epitopes, LLMGTLGIV (LV9), 1 TIHDIILECV (TV10), and KLPQLCTEL (KL9) using dextramers. A, A representative dot plot showing the %CD8 0 presence of all 3 reactivities in a tumor. B, ﬁ #1 #2 #3 #4 #5 Quanti cation of responses to all three epitopes (LV9, TV10, and KL9) from 5 HPVþ OPSCC tumors. KL9 Tumour sample

CD8

Dextramer

capacity observed for CD8/TILlow ERAP1 pairs, the ability to allotype 007 (Fig. 5D and E). These results indicate that there generate HPV-specific epitopes may be impaired preventing the is a correlation between CD8/TIL numbers and the ability to þ activation and subsequent migration and infiltration of CD8 T generate the HPV-E7 LV9 peptide epitope. Thus, the decreased cell cells into the tumor. To investigate the impact of ERAP1 on the surface expression of this HPV epitope in HPV-infected cells and generation of HPV-derived epitopes, we tested the ability of tumors from CD8/TILlow patients may compromise the ability of ERAP1 allotype combinations to generate the LV9 epitope from the immune system to recognize and kill HPV-infected cells. the minigene-encoded putative natural precursor (ED-LV9) across a range of effector:target ratios. Example results obtained from the allotype pair 001 þ 002 found in CD8/TILhigh patients (#1, 4, 5, Discussion 10), 002 þ 017 found in a CD8/TILmod patient (#13), and 007 In this study, we investigated the correlation between ERAP1 þ 011 from a CD8/TILlow patient (#18) are shown in Fig. 5A. As a allotype sequence and the amount of TILs in 22 patients with þ þ negative control, cells were transfected with the test precursor HPV OPSCC. CD8 T-cell tumor infiltration has been linked sequence together with an inactive mutant of 002 ERAP1 allo- with improved disease prognosis (5, 9). Examination of ERAP1 type (E320A). These experiments show a lower efficiency of CD8/ allotypes revealed that although many allotypes were shared TILlow-associated allotype pairs in generating LV9 from both the between the CD8/TIL groups, some differentiation between single (D-) and double (ED-) N-terminal extended precursors patients with high versus low CD8/TIL numbers was seen even compared with CD8/TILhigh, with ED-LV9 having the greatest in this small sample. Moreover, we observed that ERAP1 allotype reduction. When activity data from all allotype pairs observed pairs expressed in the high CD8/TIL group had a greater trimming were grouped according to the CD8/TIL status (Fig. 5B–E), it was capacity compared with those identified from patients with low evident that allotypes from CD8/TILlow patients were less able to CD8/TIL numbers. This was further evident from the ability of the generate LV9 from either precursor, D-LV9 or ED-LV9, than ERAP1 allotype pairs from different CD8/TIL groups to generate allotypes from CD8/TILhigh and from ED-LV9 compared with an E7 derived epitope, with low CD8/TIL ERAP1 pairs being CD8/TILmod. CD8/TILhigh pairs showed a 33% and 66% increase poorer at generating the epitope and stimulating an LV9-specific in the generation of LV9 from D-LV9 and ED-LV9 precursors, T-cell hybridoma. The anti-HPV T-cell response in patients with þ respectively (Fig. 5B–E). HPV OPSCC, a cohort linked to a better response to therapy and Examination of individual allotype pairs shows that the range prognosis (9, 26–29), is primarily directed towards antigens of responses to both precursors observed for each CD8/TIL group derived from the E6 and E7 oncoproteins (5, 7, 19). Indeed, we overlap for both CD8/TILhigh and CD8/TILmod, but not CD8/ observed responses to 3 epitopes derived from these proteins in TILlow (D-LV9 ¼ CD8/TILhigh 70%–90%, CD8/TILmod 60%–80% tumors. Thus, the reduced capacity to generate the E7 derived LV9 and CD8/TILlow 50%–60%; ED-LV9 ¼ CD8/TILhigh 60%–80%, epitope by CD8/TIL low ERAP1 pairs suggests that ERAP1 activity CD8/TILmod 55%–65%, and TILlow 40%–45%; Fig. 4D and E). In may control presentation of epitopes derived from E6 and E7 that particular, trimming of ED-LV9 showed stratification of allotype provoke antitumor T-cell responses. In addition, within each pairs based of CD8/TIL numbers (Fig. 5E). The 007 allotype has CD8/TIL group, the ERAP1 pairs identified in patients had dif- been previously reported to be a hyperactive variant (16), there- ferent peptide trimming specificities. This may explain why the fore the reduced overall trimming activity in this CD8/TILlow correlation between ERAP1 identity and CD8/TIL level is not allotype pair is likely due to the dominant negative effect of absolute.

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A High Mod Low B 800 **** ERAP1 ** allotype 600 pairs *002 + *014 *001 + *018 *001 + *002 *001 + *007 + *011 *001 + *019 *011 + *018 *011 *002 + *017 *015 + 400 E 100

K Mean AUC 200 H % Maximal LV9 response D 0 N High Mod Low Amino acid R CD8/TIL status V (X-LV9) Q 50 S G F W L I P 0

C Basic Acidic Polar uncharged 100 * 100 * 80 ** * H D * * N ** K E * Q 80 80 R 60 S 60 60 40 40 40 20 20 20

0 0 0 High Low Mod High Low Mod High Low Mod

Hydrophobic Aliphatic I Aromatic Special 60 25 50 L F * G V W % Maximal response 20 40 40 15 30

10 20 20 5 10

0 0 0 High Low Mod High Low Mod High Low Mod

Figure 4.

Amino acid specificity of ERAP1 allotype combinations generating the HPV E782-90 epitope. E1KO 293T cells were transfected with ERAP1 allotype combinations found in CD8/TILhigh, CD8/TILmoderate, and CD8/TILlow tumors, together with X-LV9 minigenes representing 15 amino acids and assessed for generation of LV9 by BE7A2Z activation. The relative presentation of trimmed X-LV9 was compared with that of the maximal response using LV9, which does not require ERAP1 activity. A, Heatmap representation of % maximal LV9 response towards each amino acid from each allotype combination. Blue represents poor trimming and red represents efficient trimming activity. B, Total trimming activity of all 15 X-LV9 substrates from each ERAP1 allotype combination grouped into CD8/TIL status and represented as AUC. C, Specificity of ERAP1 trimming activity from those found exclusively in CD8/TILhigh, CD8/TILmoderate, or CD8/TILlow tumors towards X-LV9 amino acids grouped on the basis of physicochemical properties; basic, acidic, polar uncharged, aliphatic, aromatic, and special. Data pooled from 5 independent experimental repeats SEM (, P 0.0001; , P 0.01; , P 0.05).

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A CD8/TIL high CD8/TIL mod CD8/TIL low

0.6 *001 + *002 0.6 *002 + *017 0.6 *007 + *011 LV9 LV9 D-LV9 LV9 0.4 0.4 0.4 D-LV9 ED-LV9 ED-LV9 D-LV9 0.2 0.2 0.2 BE7A2Z ED-LV9 E320A ED-LV9

response (A595) E320A ED-LV9 E320A ED-LV9 0.0 0.0 0.0 10 2 10 3 10 4 10 5 10 6 10 2 10 3 10 4 10 5 10 6 10 2 10 3 10 4 10 5 10 6 No. of transfected E1KO293T cells

B D-LV9 C ED-LV9 100 ** 100 **** ** 80 80

60 60

40 40 compared to LV9

compared to LV9 20 20 % BE7A2Z response % BE7A2Z response 0 0 High Mod Low High Mod Low D E 100 100 CD8/TILhigh mod 80 80 CD8/TIL low 60 60 CD8/TIL 40 40 20 20 compared to LV9

% BE7A2Z response 0 0 *015 *001 *015 *001 *011+*018 *007+*011 *011+*018 *007+*011 *002+*014 *001+*002 *001+*019 *001+*019 *001+*018 *002+*017 *002+*014 *001+*002 *001+*018 *002+*017

Figure 5. low Generation of HPV E782-90 from natural precursors is impaired in CD8/TIL ERAP1 allotype combinations. E1KO 293T cells were transfected with ERAP1 allotype combinations from CD8/TILhigh, CD8/TILmoderate, and CD8/TILlow or the nonfunctional active site mutant, E320A, together with minigenes encoding ﬁnal LV9 or extended precursors D-LV9 or ED-LV9 and assessed for trimming activity by the activation of BE7A2Z. A, Representative line graphs showing the trimming activity of selected ERAP1 allotype combinations from the 3 CD8/TIL groups. B and C, The relative maximum LV9 response of ERAP1 allotype combinations from each group, where each symbol represents an individual ERAP1 allotype combination transfection. Trimming activity toward a single amino acid extension (D-LV9; B) and a double amino acid extension (ED-LV9; , P 0.0001; , P 0.01; C). D and E, The relative maximum LV9 response of each individual ERAP1 allotype combination from CD8/TILhigh (black), CD8/TILmoderate (gray), and CD8/TILlow (white) toward D-LV9 (D) and ED-LV9 (E). Data pooled from three independent experimental repeats SEM.

ERAP1 polymorphism is associated with CSCC in contrast, a study revealed ERAP1 expression was upregulated in þ GWAS (12). Despite the lack of GWAS of HPV OPSCC, our head and neck squamous cell carcinoma (HNSCC) and þ observations of ERAP1 allotypes, both their sequence and HPV16 CSCC cell lines and cervical cancer (31). Although functionality, are consistent with a common immunologic the identity of the overexpressed ERAP1 allotypes was not þ mechanism between HPV OPSCC and CSCC. SNPs in ERAP1 characterized in all cell lines in that study, to assess whether together with TAP2 and LMP7 (also part of the antigen proces- they expressed SNPs associated with CSCC risk, knockdown of sing pathway) are associated with a 3-fold increase in cervical ERAP1 in 1 of 2 cervical cancer cell lines in which ERAP1 alleles carcinoma risk (12). In addition, downregulation of ERAP1 were identified resulted in increased killing by E781-91–specific expression is an independent predictor of decreased overall T cells. This cell line expressed ERAP1 that is known to survival and disease-free survival in cervical carcinoma (30). In efficiently trim peptides, but for this epitope overtrims instead,

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þ þ indicating that ERAP1 can affect the presentation of HPV- prevention of both CIN2 and CIN3 (37). Neither of these derived epitopes. Therefore, the differences in function we vaccines are effective at eliminating preexisting HPV infec- have observed between ERAP1 allotypes could be relevant for tions (38, 39) and therefore infected individuals remain at risk. controlling HPV-associated cancer. We did not examine The clearance of established HPV infections is associated with þ þ expression of the different ERAP1 allotypes in tumors in this CD4 T-helper 1 and CD8 T cells (40), with the E6 and E7 study, but altering expression of specific ERAP1 allotypes that oncoproteins targets for the response. No therapeutic vaccines are are efficient trimmers, such as 002, when coexpressed with a yet available despite several promising clinical trials (41–43). One poor trimmer, 001, within the tumor may alter the ability to contributing factor to this failure may be related to ERAP1, as the þ generate of tumor epitopes and diminish CD8 T-cell activa- expression of particular ERAP1 allotype pairs may prevent or tion. How the identity and expression of different ERAP1 reduce the capacity to generate immunogenic E6/E7 peptide allotype pairs affect generation of HPV-derived E6/E7 epitopes epitopes. Thus, affecting the ability of antigen-presenting cells to þ and their correlation with the antigen-specificity of HPV- prime HPV-specific CD8 T-cell responses in tumor draining þ specificCD8 T cells in patient cohorts, and whether these lymph node and, for T cells that traffic to the tumor, the ability correlate with positive outcome and response to checkpoint of tumor cells to activate them. Future vaccine design may need to blockade therapy, remains unclear. ERAP1 allotypes expressed incorporate the effect different ERAP1 allotypes have on the in germline DNA may provide a biomarker for prognosis generation of candidate HPV-derived epitopes. In addition, the that would guide the patient's care pathway at the time of use of ERAP1 inhibitors (44, 45) alone or in combination with diagnosis. vaccines may prevent destruction of immunogenic epitopes The mechanism by which ERAP1 trims peptides and the through overtrimming by ERAP1 allotypes (46, 47), enabling þ factors that determine the efficacy of trimming such as the activation of anti-HPV CD8 T-cell responses and tumor backbone sequence and the amino acid preceding the peptide protection. (N-1) is poorly understood. Assessment of the trimming effi- In conclusion, this study demonstrates that the function of cacy of ERAP1 containing single SNP variants showed that the expressed ERAP1 allotypes correlates with the amount of infil- catalytic rate of the variant enzymes was affected to a similar trating lymphocytes in tumors of patients with OPSCC and thus, degree for a number of peptide substrates with different back- prognosis. CD8/TILlow ERAP1 allotype pairs were poor at gener- bones (32). The identity of the N-1 amino acid, however, ating an HPV E7 epitope, indicating that the lack of T-cell influenced the ability of ERAP1 to generate the final peptide infiltration and subsequent poorer antitumor response may be epitope (11, 33) suggesting that the N-1 amino acid affects related to the ability of HPV-infected/tumor cells to generate HPV- trimming to a greater extent than the backbone. Consistent specific peptide epitopes. with this, we found that for 2 different peptide backbones, SHL8 and LV9 (which bind to different MHC molecules), the Disclosure of Potential Conflicts of Interest general trimming activity of the different ERAP1 pairs was T. Elliott is a consultant/advisory board member of Avvinity Therapeutics. similar. However, we found that the precise amino acid trim- No potential conflicts of interest were disclosed by the other authors. ming specificity profiles of ERAP1 allotypes/pairs were different between X-SHL8 and X-LV9. For example, the "top" 3 specificities for the single allotype 015 (CD8/TILmod)wereE,H,and Authors' Contributions Q for X-SHL8 and E, H, and K for X-LV9. For single allotype Conception and design: E. Reeves, E.V. King, T. Elliott, E. James low Development of methodology: E. Reeves, O. Wood, G.J. Thomas, T. Elliott, 001 (CD8/TIL ), we observed a greater difference where the E. James fi "top" 3 speci cities were C, A, and E for X-SHL8, but E, K, and N Acquisition of data (provided animals, acquired and managed patients, for X-LV9. These differences indicate that compared with over- provided facilities, etc.): E. Reeves, O. Wood, C.H. Ottensmeier, E.V. King, all trimming phenotype, amino acid specificity profiles may G.J. Thomas depend on the peptide backbone. A greater understanding of Analysis and interpretation of data (e.g., statistical analysis, biostatistics, how these specificity profiles relate to the core peptide sequence computational analysis): E. Reeves, T. Elliott, E. James Writing, review, and/or revision of the manuscript: E. Reeves, could enable the development of ERAP1 processing algorithms C.H. Ottensmeier, E.V. King, G.J. Thomas, T. Elliott, E. James for individual and combined allotypes. This could be integrated Administrative, technical, or material support (i.e., reporting or organizing with algorithms that predict proteasome cleavage (20–22), data, constructing databases): E. James peptide binding affinity (34), and the effects of peptide abun- Study supervision: T. Elliott, E. James dance and intracellular competition (35) to improve the ability to predict whether candidate peptides are likely to be presented Acknowledgments fi at the cell surface in suf cient abundance to prime an effective The authors would like to thank Pandurangan Vijayanand for his support in CTL response. collating samples and Nasia Kontouli for technical support. This work was ERAP2, a homolog of ERAP1 that is less polymorphic, may supported by Cancer Research UK Programme Grant A16997 awarded to also play a role in the generation of HPV-derived epitopes. E. James and T. Elliott. ERAP2 has altered substrate handling and amino acid trimming preferences compared with ERAP1, showing a preference for The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Arg (R) and less enzymatic activity (36). Despite its reduced advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate activity, ERAP2 likely also contributes to the generation of HPV- this fact. derived epitopes. Currently, three commercially available prophylactic HPV vac- Received July 23, 2018; revised February 15, 2019; accepted May 9, 2019; cines target high-risk HPV types (37). These vary in efficacy for published first May 31, 2019.

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www.aacrjournals.org Cancer Immunol Res; 7(7) July 2019 1213

HPV Epitope Processing Differences Correlate with ERAP1 Allotype and Extent of CD8 + T-cell Tumor Infiltration in OPSCC

Emma Reeves, Oliver Wood, Christian H. Ottensmeier, et al.

Cancer Immunol Res 2019;7:1202-1213. Published OnlineFirst May 31, 2019.

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