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

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

Immune responses against mutated ligands in PBMC of patient Mel15.Clinical course and retrieval of patient material (a). Schematic overview of the experimental design of recall immune responses among blood-derived T cells from patient Mel15 (b). Early immune responses detected in PBMC derived from different blood withdrawals two days after in-vitro peptide stimulation (c). Time course of specific reactivities of blood-derived PBMC obtained at different time points against the eight identified mutated epitopes from patient Mel15. All analyses were performed in duplicates and spot counts were adjusted to 104 cells (d). Intracellular cytokine staining (ICS) of an expanded NCAPG2P333L specific T-cell line from day 546 (PBMC-NCAPG2-546) after co-incubation with peptide pulsed T2-A3 target cells for 5 h (e). ICS of T-cell line PBMC-SYTL4-740 stimulated with SYTL4S363F from day 740 after co-culture with peptide pulsed T2-B27 target cells (f). Staining of line PBMC-NCAPG2-546 with the specific multimer in comparison to irrelevant multimer staining (g) IFN-g secretion after coincubation of T-cell clone PBMC-SYTL4clone1 derived from line PBMC-SYTL4-740 with peptide pulsed and minigene-transduced LCL1 (results of triplicates) (h). Data from experiments with triplicates are shown as mean±s.d., data resulting from duplicates are shown as mean.
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f5: Immune responses against mutated ligands in PBMC of patient Mel15.Clinical course and retrieval of patient material (a). Schematic overview of the experimental design of recall immune responses among blood-derived T cells from patient Mel15 (b). Early immune responses detected in PBMC derived from different blood withdrawals two days after in-vitro peptide stimulation (c). Time course of specific reactivities of blood-derived PBMC obtained at different time points against the eight identified mutated epitopes from patient Mel15. All analyses were performed in duplicates and spot counts were adjusted to 104 cells (d). Intracellular cytokine staining (ICS) of an expanded NCAPG2P333L specific T-cell line from day 546 (PBMC-NCAPG2-546) after co-incubation with peptide pulsed T2-A3 target cells for 5 h (e). ICS of T-cell line PBMC-SYTL4-740 stimulated with SYTL4S363F from day 740 after co-culture with peptide pulsed T2-B27 target cells (f). Staining of line PBMC-NCAPG2-546 with the specific multimer in comparison to irrelevant multimer staining (g) IFN-g secretion after coincubation of T-cell clone PBMC-SYTL4clone1 derived from line PBMC-SYTL4-740 with peptide pulsed and minigene-transduced LCL1 (results of triplicates) (h). Data from experiments with triplicates are shown as mean±s.d., data resulting from duplicates are shown as mean.

Mentions: We next asked if the MS-detected mutated peptide ligands represent neoepitopes that can be recognized by the patient's own T cells. We selected patient Mel15 as for this patient diverse mutated peptide ligands were identified and miscellaneous biomaterial could be collected. The detailed clinical course including biomaterial collection of the patient is shown in Fig. 5a. For the investigation of recall responses, we stimulated unfractionated PBMC derived from diverse venipunctures in the course of the disease following application of Ipilimumab (Fig. 5a,b). Without any further enrichment, we identified defined T-cell responses by ELISpot as early as two days after stimulation of PBMC (Fig. 5c). Notably, specific responses were repeatedly observed against SYTL4S363F at that early time point (Fig. 5c). Prolonged peptide stimulation of PBMC derived from diverse blood venipunctures resulted in expansion of T cells with specificity for SYTL4S363F, as well as NCAPG2P333L but not for other peptides (Fig. 5d). Dynamic courses of specific responses observed against these two peptides indicate a decline of specific T-cell responses over time. The quality of T-cell responses against SYTL4S363F was superior compared with NCAPG2P333L as shown by higher frequencies of T cells with dual cytokine secretion (Fig. 5e,f). Of note, wt peptides were not recognized (Fig. 5e,f). Specificity of defined T-cell lines was further confirmed by multimer staining of NCAPG2P333L–specific T cells (Fig. 5g). In case of T-cell line PBMC-SYTL4-740, we were able to isolate a specific clone, PBMC-SYTL4clone1, which recognized endogenously processed mutated but not wt peptide after minigene transfer of respective gene sequences of SYTL4 (Fig. 5h).


Direct identification of clinically relevant neoepitopes presented on native human melanoma tissue by mass spectrometry
Immune responses against mutated ligands in PBMC of patient Mel15.Clinical course and retrieval of patient material (a). Schematic overview of the experimental design of recall immune responses among blood-derived T cells from patient Mel15 (b). Early immune responses detected in PBMC derived from different blood withdrawals two days after in-vitro peptide stimulation (c). Time course of specific reactivities of blood-derived PBMC obtained at different time points against the eight identified mutated epitopes from patient Mel15. All analyses were performed in duplicates and spot counts were adjusted to 104 cells (d). Intracellular cytokine staining (ICS) of an expanded NCAPG2P333L specific T-cell line from day 546 (PBMC-NCAPG2-546) after co-incubation with peptide pulsed T2-A3 target cells for 5 h (e). ICS of T-cell line PBMC-SYTL4-740 stimulated with SYTL4S363F from day 740 after co-culture with peptide pulsed T2-B27 target cells (f). Staining of line PBMC-NCAPG2-546 with the specific multimer in comparison to irrelevant multimer staining (g) IFN-g secretion after coincubation of T-cell clone PBMC-SYTL4clone1 derived from line PBMC-SYTL4-740 with peptide pulsed and minigene-transduced LCL1 (results of triplicates) (h). Data from experiments with triplicates are shown as mean±s.d., data resulting from duplicates are shown as mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5121339&req=5

f5: Immune responses against mutated ligands in PBMC of patient Mel15.Clinical course and retrieval of patient material (a). Schematic overview of the experimental design of recall immune responses among blood-derived T cells from patient Mel15 (b). Early immune responses detected in PBMC derived from different blood withdrawals two days after in-vitro peptide stimulation (c). Time course of specific reactivities of blood-derived PBMC obtained at different time points against the eight identified mutated epitopes from patient Mel15. All analyses were performed in duplicates and spot counts were adjusted to 104 cells (d). Intracellular cytokine staining (ICS) of an expanded NCAPG2P333L specific T-cell line from day 546 (PBMC-NCAPG2-546) after co-incubation with peptide pulsed T2-A3 target cells for 5 h (e). ICS of T-cell line PBMC-SYTL4-740 stimulated with SYTL4S363F from day 740 after co-culture with peptide pulsed T2-B27 target cells (f). Staining of line PBMC-NCAPG2-546 with the specific multimer in comparison to irrelevant multimer staining (g) IFN-g secretion after coincubation of T-cell clone PBMC-SYTL4clone1 derived from line PBMC-SYTL4-740 with peptide pulsed and minigene-transduced LCL1 (results of triplicates) (h). Data from experiments with triplicates are shown as mean±s.d., data resulting from duplicates are shown as mean.
Mentions: We next asked if the MS-detected mutated peptide ligands represent neoepitopes that can be recognized by the patient's own T cells. We selected patient Mel15 as for this patient diverse mutated peptide ligands were identified and miscellaneous biomaterial could be collected. The detailed clinical course including biomaterial collection of the patient is shown in Fig. 5a. For the investigation of recall responses, we stimulated unfractionated PBMC derived from diverse venipunctures in the course of the disease following application of Ipilimumab (Fig. 5a,b). Without any further enrichment, we identified defined T-cell responses by ELISpot as early as two days after stimulation of PBMC (Fig. 5c). Notably, specific responses were repeatedly observed against SYTL4S363F at that early time point (Fig. 5c). Prolonged peptide stimulation of PBMC derived from diverse blood venipunctures resulted in expansion of T cells with specificity for SYTL4S363F, as well as NCAPG2P333L but not for other peptides (Fig. 5d). Dynamic courses of specific responses observed against these two peptides indicate a decline of specific T-cell responses over time. The quality of T-cell responses against SYTL4S363F was superior compared with NCAPG2P333L as shown by higher frequencies of T cells with dual cytokine secretion (Fig. 5e,f). Of note, wt peptides were not recognized (Fig. 5e,f). Specificity of defined T-cell lines was further confirmed by multimer staining of NCAPG2P333L–specific T cells (Fig. 5g). In case of T-cell line PBMC-SYTL4-740, we were able to isolate a specific clone, PBMC-SYTL4clone1, which recognized endogenously processed mutated but not wt peptide after minigene transfer of respective gene sequences of SYTL4 (Fig. 5h).

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