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

Abstract

Presence of tumor-infiltrating lymphocytes (TIL) predicts survival in many cancer types. In HPV-driven cancers, cervical and oropharyngeal squamous cell carcinomas (CSCC and OPSCC, respectively), numbers of infiltrating T cells, particularly CD8⁺ T cells, and presentation of HPV E6/E7 epitopes are associated with improved prognosis. Endoplasmic reticulum aminopeptidase 1 (ERAP1) regulates the presented peptide repertoire, trimming peptide precursors prior to MHC I loading. ERAP1 is polymorphic, and allotypic variation of ERAP1 enzyme activity has an impact on the presented peptide repertoire. Individual SNPs are associated with incidence and outcome in a number of diseases, including CSCC. Here, we highlight the requirement for ERAP1 in the generation of HPV E6/E7 epitopes and show that the functional activity of ERAP1 allotype combinations identified in OPSCC correlate with tumor-infiltrating CD8⁺ T-cell (CD8)/TIL (CD8/TIL) status of the tumor. Functional analyses revealed that ERAP1 allotype combinations associated with CD8/TIL^low tumors have a reduced capacity to generate both a model antigen SIINFEHL and the HPV-16 E7_82-90 epitope LLMGTLGIV from N-terminally extended precursor peptides. In contrast, ERAP1 allotypes from CD8/TIL^high tumors generated the epitopes efficiently. These data reveal that ERAP1 function correlates with CD8/TIL numbers and, by implication, prognosis, suggesting that the presentation of HPV-16 epitopes at the cell surface, resulting in an anti-HPV T-cell response, may depend on the ERAP1 allotype combinations expressed within an individual.

Figure 1. N-terminal amino acid trimming specificity by OPSCC ERAP1 allotype combinations.

E1KO 293T cells were transfected with ERAP1 allotype combinations from OPSCC patients together with H2-K^b 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 panels (A) CD8/TIL^high, (B) CD8/TIL^moderate, (C) CD8/TIL^Low, (D) CD8/TIL^high and CD8/TIL^moderate, (E) CD8/TIL^high and CD8/TIL^low and (F) CD8/TIL^moderate and CD8/TIL^low. The relative presentation of trimmed X-SHL8 was compared to that of the maximal response using SHL8, which does not require ERAP1 activity. Data pooled from four independent experimental repeats ±SEM.

Figure 2. ERAP1 allotype combinations from CD8/TIL^low 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 area under curve (AUC). (B) Table showing both the individual AUC for allotype pairs in each CD8/TIL group as well as the mean for each group. (C) Specificity of ERAP1 trimming activity from those found exclusively in CD8/TIL^high, CD8/TIL^moderate or CD8/TIL^low 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 four independent experimental repeats ±SEM (***p = <0.001; **p = <0.01; *p = <0.05).

Figure 3. Presence of anti-HPV E6/E7 epitope CD8⁺ T cell responses in HPV⁺ OPSCC tumors.

Tumors from HPV⁺ OPSCC patients were assessed for the presence of CD8⁺ T cell responses to the HLA-A*0201 restricted HPV E6/E7 epitopes, LLMGTLGIV (LV9), TIHDIILECV (TV10), and KLPQLCTEL (KL9) using dextramers. (A) A representative dot plot showing the presence of all three reactivities in a tumor. (B) Quantification of responses to all three epitopes (LV9, TV10 and KL9) from five HPV⁺ OPSCC tumors.

Figure 4. Amino acid specificity of ERAP1 allotype combinations generating the HPV E7_82-90 epitope.

E1KO 293T cells were transfected with ERAP1 allotype combinations found in CD8/TIL^high, CD8/TIL^moderate and CD8/TIL^low 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 to that of the maximal response using LV9, which does not require ERAP1 activity. (A) Heat map 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 area under curve (AUC). (C) Specificity of ERAP1 trimming activity from those found exclusively in CD8/TIL^high, CD8/TIL^moderate or CD8/TIL^low tumors towards X-LV9 amino acids grouped based on physicochemical properties; basic, acidic, polar uncharged, aliphatic, aromatic and special. Data pooled from five independent experimental repeats ±SEM (****p = <0.0001; **p = <0.01; *p = <0.05).

Figure 5. Generation of HPV E7_82-90 from natural precursors is impaired in CD8/TIL^low ERAP1 allotype combinations.

E1KO 293T cells were transfected with ERAP1 allotype combinations from CD8/TIL^high, CD8/TIL^moderate and CD8/TIL^low or the non-functional 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 three 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. (B) Trimming activity towards a single amino acid extension (D-LV9) and (C) a double amino acid extension (ED-LV9; **** p = <0.0001; ** p = <0.01). (D and E) The relative maximum LV9 response of each individual ERAP1 allotype combination from CD8/TIL^high (black), CD8/TIL^moderate (grey), and CD8/TIL^low (white) towards D-LV9 (D) and ED-LV9 (E). Data pooled from three independent experimental repeats ±SEM.