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Direct identification of clinically relevant neoepitopes presented on native human melanoma tissue by mass spectrometry

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ABSTRACT

Although mutations may represent attractive targets for immunotherapy, direct identification of mutated peptide ligands isolated from human leucocyte antigens (HLA) on the surface of native tumour tissue has so far not been successful. Using advanced mass spectrometry (MS) analysis, we survey the melanoma-associated immunopeptidome to a depth of 95,500 patient-presented peptides. We thereby discover a large spectrum of attractive target antigen candidates including cancer testis antigens and phosphopeptides. Most importantly, we identify peptide ligands presented on native tumour tissue samples harbouring somatic mutations. Four of eleven mutated ligands prove to be immunogenic by neoantigen-specific T-cell responses. Moreover, tumour-reactive T cells with specificity for selected neoantigens identified by MS are detected in the patient's tumour and peripheral blood. We conclude that direct identification of mutated peptide ligands from primary tumour material by MS is possible and yields true neoepitopes with high relevance for immunotherapeutic strategies in cancer.

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Characterization of mutant-specific T-cell responses in HLA-matched healthy donors.T-cell responses of two different matched healthy donors against neoepitopes AKAP6M1482I and NOP16P169L. Effector cells were coincubated in duplicates with T2-A3 or T2-B7 pulsed either with the relevant peptide or control peptides with the same HLA restriction as the mutated ligands, results are shown as mean (a). Staining of T-cell line HD1-AKAP6 with the mutated or wt multimer (b). IFN-g release of the T-cell line on peptide titration of AKAP6M1482I and its non-mutated counterpart using T2-A3 as targets (duplicates are depicted as mean) (c). IFN-g secretion (left Y-axis) and target-cell lysis (right Y-axis) after coincubation of the T-cell line HD1-AKAP6 with peptide-pulsed and minigene-transduced LCL1 cells performed in triplicates, data shown as mean±s.d. (d). Intracellular cytokine staining (IFN-g, TNF-a and IL-2) on co-culture of the T-cell line HD1-AKAP6 with LCL1 cells, either peptide-pulsed or minigene-transduced, determined by flow cytometry. Cells were gated on ethidium monoazide bromide-negative and CD8-positive events (e).
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f7: Characterization of mutant-specific T-cell responses in HLA-matched healthy donors.T-cell responses of two different matched healthy donors against neoepitopes AKAP6M1482I and NOP16P169L. Effector cells were coincubated in duplicates with T2-A3 or T2-B7 pulsed either with the relevant peptide or control peptides with the same HLA restriction as the mutated ligands, results are shown as mean (a). Staining of T-cell line HD1-AKAP6 with the mutated or wt multimer (b). IFN-g release of the T-cell line on peptide titration of AKAP6M1482I and its non-mutated counterpart using T2-A3 as targets (duplicates are depicted as mean) (c). IFN-g secretion (left Y-axis) and target-cell lysis (right Y-axis) after coincubation of the T-cell line HD1-AKAP6 with peptide-pulsed and minigene-transduced LCL1 cells performed in triplicates, data shown as mean±s.d. (d). Intracellular cytokine staining (IFN-g, TNF-a and IL-2) on co-culture of the T-cell line HD1-AKAP6 with LCL1 cells, either peptide-pulsed or minigene-transduced, determined by flow cytometry. Cells were gated on ethidium monoazide bromide-negative and CD8-positive events (e).

Mentions: To investigate if mutated peptide ligands may be immunogenic in matched healthy donors, we stimulated naïve T cells isolated from different donors with mutated peptide ligands. We identified additional reactivity against two peptides, AKAP6M1482I derived from Mel15 and NOP16P169L derived from Mel8 (Fig. 7a). An expanded T-cell line, HD1-AKAP6, with specificity for AKAP6M1482I was further characterized. We observed specific binding of respective multimer but not wt multimer (Fig. 7b). In contrast, peptide titration experiments showed recognition of the mutant but also wt peptide, the latter with reduced functional avidity (Fig. 7c). Functional quality of T-cell responses against wt and mutated peptides were additionally investigated in detail with respect to heterogeneous cytokine release and cytotoxicity (Fig. 7d,e). Therefore, target cells either pulsed with defined peptides or transduced with minigenes were used. Of note, cytokine responses against wt peptide were inferior when compared with the mutated counterpart whereas the cytotoxic responses were comparable (Fig. 7d,e).


Direct identification of clinically relevant neoepitopes presented on native human melanoma tissue by mass spectrometry
Characterization of mutant-specific T-cell responses in HLA-matched healthy donors.T-cell responses of two different matched healthy donors against neoepitopes AKAP6M1482I and NOP16P169L. Effector cells were coincubated in duplicates with T2-A3 or T2-B7 pulsed either with the relevant peptide or control peptides with the same HLA restriction as the mutated ligands, results are shown as mean (a). Staining of T-cell line HD1-AKAP6 with the mutated or wt multimer (b). IFN-g release of the T-cell line on peptide titration of AKAP6M1482I and its non-mutated counterpart using T2-A3 as targets (duplicates are depicted as mean) (c). IFN-g secretion (left Y-axis) and target-cell lysis (right Y-axis) after coincubation of the T-cell line HD1-AKAP6 with peptide-pulsed and minigene-transduced LCL1 cells performed in triplicates, data shown as mean±s.d. (d). Intracellular cytokine staining (IFN-g, TNF-a and IL-2) on co-culture of the T-cell line HD1-AKAP6 with LCL1 cells, either peptide-pulsed or minigene-transduced, determined by flow cytometry. Cells were gated on ethidium monoazide bromide-negative and CD8-positive events (e).
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f7: Characterization of mutant-specific T-cell responses in HLA-matched healthy donors.T-cell responses of two different matched healthy donors against neoepitopes AKAP6M1482I and NOP16P169L. Effector cells were coincubated in duplicates with T2-A3 or T2-B7 pulsed either with the relevant peptide or control peptides with the same HLA restriction as the mutated ligands, results are shown as mean (a). Staining of T-cell line HD1-AKAP6 with the mutated or wt multimer (b). IFN-g release of the T-cell line on peptide titration of AKAP6M1482I and its non-mutated counterpart using T2-A3 as targets (duplicates are depicted as mean) (c). IFN-g secretion (left Y-axis) and target-cell lysis (right Y-axis) after coincubation of the T-cell line HD1-AKAP6 with peptide-pulsed and minigene-transduced LCL1 cells performed in triplicates, data shown as mean±s.d. (d). Intracellular cytokine staining (IFN-g, TNF-a and IL-2) on co-culture of the T-cell line HD1-AKAP6 with LCL1 cells, either peptide-pulsed or minigene-transduced, determined by flow cytometry. Cells were gated on ethidium monoazide bromide-negative and CD8-positive events (e).
Mentions: To investigate if mutated peptide ligands may be immunogenic in matched healthy donors, we stimulated naïve T cells isolated from different donors with mutated peptide ligands. We identified additional reactivity against two peptides, AKAP6M1482I derived from Mel15 and NOP16P169L derived from Mel8 (Fig. 7a). An expanded T-cell line, HD1-AKAP6, with specificity for AKAP6M1482I was further characterized. We observed specific binding of respective multimer but not wt multimer (Fig. 7b). In contrast, peptide titration experiments showed recognition of the mutant but also wt peptide, the latter with reduced functional avidity (Fig. 7c). Functional quality of T-cell responses against wt and mutated peptides were additionally investigated in detail with respect to heterogeneous cytokine release and cytotoxicity (Fig. 7d,e). Therefore, target cells either pulsed with defined peptides or transduced with minigenes were used. Of note, cytokine responses against wt peptide were inferior when compared with the mutated counterpart whereas the cytotoxic responses were comparable (Fig. 7d,e).

View Article: PubMed Central - PubMed

ABSTRACT

Although mutations may represent attractive targets for immunotherapy, direct identification of mutated peptide ligands isolated from human leucocyte antigens (HLA) on the surface of native tumour tissue has so far not been successful. Using advanced mass spectrometry (MS) analysis, we survey the melanoma-associated immunopeptidome to a depth of 95,500 patient-presented peptides. We thereby discover a large spectrum of attractive target antigen candidates including cancer testis antigens and phosphopeptides. Most importantly, we identify peptide ligands presented on native tumour tissue samples harbouring somatic mutations. Four of eleven mutated ligands prove to be immunogenic by neoantigen-specific T-cell responses. Moreover, tumour-reactive T cells with specificity for selected neoantigens identified by MS are detected in the patient's tumour and peripheral blood. We conclude that direct identification of mutated peptide ligands from primary tumour material by MS is possible and yields true neoepitopes with high relevance for immunotherapeutic strategies in cancer.

No MeSH data available.


Related in: MedlinePlus