Novel H-2Db-restricted CD8 epitope derived from mouse MAGE-type antigen P1A mediates antitumor immunity in C57BL/6 mice

Abstract

Background: Melanoma antigen gene (MAGE)-type antigens are promising targets for cancer immunotherapy as they are expressed in cancer cells but not in normal tissues, except for male germline cells. The mouse P1A antigen shares this MAGE-type expression pattern and has been used as a target antigen in preclinical tumor models aiming to induce antitumor CD8⁺ T-cell responses. However, so far only one MHC I-restricted P1A epitope has been identified. It is presented by H-2L^d in mice of the H-2^d genetic background such as DBA/2 and BALB/c. Given the availability of multiple genetically altered strains of mice in the C57BL/6 background, it would be useful to define P1A T-cell epitopes presented by the H-2^b haplotype, to facilitate more refined mechanistic studies.

Methods: We employed a heterologous prime-boost vaccination strategy based on a chimpanzee adenovirus (ChAdOx1) and a modified vaccinia Ankara (MVA) encoding P1A, to induce P1A-specific T-cell responses in C57BL/6 mice. Vaccine-induced responses were measured by intracellular cytokine staining and multiparameter flow cytometry. We mapped the immunogenic CD8 epitope and cloned the cognate T-cell receptor (TCR), which we used for adoptive cell therapy.

Results: ChAdOx1/MVA-P1A vaccination induces a strong P1A-specific CD8⁺ T-cell response in C57BL/6 mice. This response is directed against a single 9-amino acid peptide with sequence FAVVTTSFL, corresponding to P1A amino acids 43-51. It is presented by H-2D^b. P1A vaccination, especially with ChAdOx1 administered intramuscularly and MVA delivered intravenously, protected mice against P1A-expressing EL4 (EL4.P1A) tumor cell challenge. We identified and cloned four TCRs that are specific for the H-2D^b-restricted P1A_43-51 peptide. T cells transduced with these TCRs recognized EL4.P1A but not MC38.P1A and B16F10.P1A tumor cells, likely due to differences in the proteasome subtypes present in these cells. Adoptive transfer of these T cells in mice bearing EL4.P1A tumors reduced tumor growth and increased survival.

Conclusions: We identified the first CD8⁺ T-cell epitope of the MAGE-type P1A tumor antigen presented in the H-2^b background. This opens new perspectives for mechanistic studies dissecting MAGE-type specific antitumor immunity, making use of the wealth of genetically altered mouse strains available in the C57BL/6 background. This should facilitate the advancement of specific cancer immunotherapies.

Keywords: Adoptive cell therapy - ACT; Major histocompatibility complex - MHC; T cell; T cell Receptor - TCR; Vaccine.

Figure 1. ChAdOx1/MVA-P1A vaccination induces a strong P1A-specific CD8⁺ T cell response in C57BL/6 mice. C57BL/6 mice were vaccinated intramuscularly with ChAdOx1 prime and MVA boost expressing P1A or DPY (irrelevant antigen) or PBS at different doses and time intervals. (A) Experimental scheme. (B) Ex vivo IFN-γ production by CD8⁺ T cells in the peripheral blood on stimulation with P1A peptide pools detected by intracellular cytokine staining (ICS) and flow cytometry in the different treatment groups. ChAdOx1-P1A at 5×10⁸ IU prime and MVA-P1A at 10⁷ PFU boost were given 4 weeks apart and blood sampling was performed 2 weeks postprime and 2 weeks postboost. (C) Representative dot plot of IFN-γ production within the CD8 and CD4 populations of an individual mouse; (D) IFN-γ⁺, TNF-α⁺ and IL-2⁺ percentage of CD8⁺ T cells in all mice; (E) polyfunctionality of responding CD8⁺ T cells are represented as pie charts of mean relative proportions. Data are shown as mean±SEM, each symbol represents an individual mouse, 5–10 mice per group with key groups pooled from 2 to 3 experiments. Statistical analysis: Kruskal-Wallis with Dunn’s multiple comparisons (B), two-way ANOVA (D); *p<0.05, **p<0.01, ****p<0.0001. ANOVA, analysis of variance; IU, infectious unit; MVA, modified vaccinia Ankara; PFU, plaque forming unit.

Figure 2. Epitope mapping of the P1A protein in C57BL/6 mice. Spleens from vaccinated C57BL/6 mice (ChAdOx1-P1A 5×10⁸ IU prime and MVA-P1A 10⁷ PFU boost) were harvested, processed to single-cell suspension and stimulated with different peptide fractions of the P1A protein or vehicle control (DMSO). Frequency of IFN-γ⁺ cells in CD8⁺ T cells detected by ICS on: (A) stimulation with overlapping 15-mer P1A peptides organized in five sub-pools or all peptides covering the entire protein; (B) stimulation with all individual 15-mers contained in Sub-pool 1. (C) Net-MHC 4.0 peptide binding prediction of the top five ranked P1A peptides in H-2^b background within the amino acid interval 37–55. (D) Proportion of IFN-γ⁺ cells in CD8 detected by ICS on stimulation with previously determined immunogenic intervals or their adjacent peptides, and the first-ranked predicted nonameric epitope FAVVTTSLF P1A_43-51. (E) IFN-γ-producing cells in response to two 8-mer generated as truncated versions of P1A_43-51-FAVVTTSFL: FAVVTTSF or AVVTTSFL. (F) Graphical representation of the P1A protein and the two immunogenic epitopes in H2^d mice (blue) or H2^b mice (orange), with one shared amino acid (purple). Data are shown as mean+SEM, each symbol represents an individual mouse, n=3. Statistical analysis: Friedman test with Dunn’s multiple comparisons, *p≤0.05. ICS, intracellular cytokine staining; IU, infectious unit.

Figure 3. Identification of P1A_(43-51)-specific TCR sequences. (A) Experimental scheme. Spleens from ChAdOx1/MVA-P1A vaccinated mice were harvested and processed to single-cell suspension. P1A-specific CD8⁺ T cells were labeled with an H-2D^b P1A_43-51 tetramer and isolated by FACS. cDNA libraries were prepared from P1A-specific CD8⁺ single cells; RNA from 1252 cells was sequenced. (B) Representative dot plot of H-2D^b P1A_43-51 tetramer⁺ CD8⁺ T cells sorted by FACS. (C) V(D)J gene sequences for TCR-α and TCR-β chain were determined and stratified by their frequency of total sequenced barcodes. (D) Four of the most represented clonotypes (A–D) were cloned into pMIG-II retroviral vectors to transduce CD8⁺ splenocytes; TCR transduction efficiency was detected by GFP expression compared with mock untransduced cells. (E) Representative flow cytometry plots of IFN-γ expression by TCR-transduced CD8⁺ T cells cocultured with either P1A^- EL4 wild-type (WT), EL4.P1A cells or peptide-pulsed EL4.P1A cells, or stimulated with soluble P1A_43-51 peptide or DMSO as positive and negative controls, respectively. (F) Magnitude of IFN-γ expression by TCR-transduced CD8⁺ T cells, pooled experiments (n=3, duplicates) shown relative to P1A_43-51 peptide positive control. (G) P1A-specific T-cell responses on stimulation with endogenous or peptide-pulsed P1A+solid tumors (MC38.P1A and B16F10.P1A) compared with the parental P1A-WT lines, shown as frequency of IFN-γ⁺ CD8⁺ cells relative to P1A peptide control. (H) Representative western blot to assess the presence of proteasome inducible subunits (β1i, β2i and β5i) within the C57BL/6 tumor lines EL4, MC38 and B16F10, compared with T429 cells engineered to contain only a single proteasome subtype, as indicated (SP (β1-β2 -β5); SIP (β1β2β5i): DIP (β1iβ2β5i); IP (β1iβ2iβ5i). Statistical analysis: RM one-way ANOVA with Tukey’s multiple comparisons, *p≤0.05. ANOVA, analysis of variance; DIP, double intermediate proteasome; IP, immunoproteasome; SIP, single-intermediate proteasome; SP, standard proteasome.

Figure 4. Prophylactic P1A vaccination improves tumor control in C57BL/6 mice. C57BL/6 mice were vaccinated with 5×10⁸ IU ChAdOx1-P1A and 10⁷ PFU MVA-P1A 4 weeks apart via either intramuscular or intravenous routes, or with ChAdOx1-DPY (intramuscular) and MVA-DPY (intravenous) irrelevant antigen control or PBS sham. Mice were then implanted with EL4.P1A 0.2×10⁶ cells subcutaneously in the right flank 2 weeks post boost. (A) Experimental scheme. (B) Vaccine immunogenicity was assessed through IFN-γ⁺ CD8⁺ T cells detected by ICS on ex vivo P1A peptide stimulation of the peripheral PBMCs, 14–17 days after prime (circle) and 10–13 days after boost (diamond); data representative of 2–4 experiments. (C) Tumor volume was measured, shown as mean±SEM; (D) tumor volume at day 17 for individual mice; (E) overall survival following tumor implantation; (F) individual tumor growth curves for each mouse in the treatment group. (G) Correlation analysis of day 17 tumor volume and post-boost CD8⁺ IFN-γ⁺ frequency for each vaccinated mouse; lines summarize correlation by vaccination route. Pooled data from 2 to 4 experiments of n=6–12 mice per group (C–G). Bars represent mean (B, D). Statistical analysis: Kruskal-Wallis with Dunn’s multiple comparisons (B, D) Two-way ANOVA with Tukey’s post hoc multiple comparisons (C), log-rank Mantel-Cox test (E) and Spearman rank correlation (G). *p<0.05, **p<0.01, ****p<0.0001. ANOVA, analysis of variance; ICS, intracellular cytokine staining; MVA, modified vaccinia Ankara; PBMCs, peripheral blood mononuclear cell.

Figure 5. Adoptive cell transfer of P1A_43-51 TCR-transduced CD8⁺ T cells exhibits good therapeutic control of EL4.P1A tumor growth in C57BL/6 mice. (A) Experimental scheme: C57BL/6 mice were implanted with 0.2×10⁶ EL4.P1A cells subcutaneously, before receiving intramuscular vaccination with alternating ChAdOx1-P1A (5×10⁷ IU) and MVA-P1A (5×10⁶ PFU) at 1-week intervals, for the duration of the study; (B) mean tumor volume and (C) survival were recorded. Data pooled from two experiments, 5–6 mice/group. (D) C57BL/6 mice were implanted with 0.2×10⁶ EL4.P1A cells subcutaneously before receiving 10⁷ TCR-transduced CD8⁺ cells (TCR A, TCR B or OT-I irrelevant TCR control or PBS) administered intravenously on day 7 following tumor implantation. (E) Mean tumor growth, (F) survival and (G) individual tumor growth curves are shown. (H) Proportion of IFN-γ⁺ CD8⁺ T cells detected by ICS following ex vivo P1A-specific stimulation of the PBMCs 10 days post-ACT. Bars represent the mean. (I) Correlation between tumor size (day 15) and CD8⁺ IFN-γ⁺ (day 17). Data pooled from two experiments, symbols represent individual mice, n=8–12 mice per group. Statistical analysis: Ordinary two-way ANOVA (B); two-way ANOVA with Dunnett’s multiple comparisons (E); log-rank Mantel-Cox test (C, F); Kruskal-Wallis with Dunn’s multiple comparisons (H) and Spearman rank correlation (I). *p<0.05, **p<0.01, ****p<0.0001. ACT, adoptive cell transfer; ANOVA, analysis of variance; IM, intramuscular; MVA, modified vaccinia Ankara; PBMC, peripheral blood mononuclear cell.