Spliced peptides and cytokine driven changes in the immunopeptidome of melanoma

Abstract

SummaryAntigen-recognition by CD8+ T cells is governed by the pool of peptide antigens presented on the cell surface in the context of HLA class I complexes. Recent studies have shown not only a high degree of plasticity in the immunopeptidome, but also that a considerable fraction of all presented peptides is generated through proteasome-mediated splicing of non-contiguous regions of proteins to form novel peptide antigens. Here we used high-resolution mass-spectrometry combined with new bioinformatic approaches to characterize the immunopeptidome of melanoma cells in the presence or absence of interferon-γ. In total, we identified more than 60,000 peptides from a single patient derived cell line (LM-MEL-44) and demonstrated that interferon-γ induced marked changes in the peptidome with an overlap of only ∼50% between basal and treated cells. Around 6-8% of the peptides were identified as cis-spliced peptides, and 2213 peptides (1827 linear, 386 cis-spliced peptides) were derived from known melanoma-associated antigens. These peptide antigens were equally distributed between the constitutive and interferon-γ induced peptidome. We next examined additional HLA-matched patient derived cell lines to investigate how frequently these peptides were identified and found that a high proportion of both linear and spliced peptides were conserved between individual patient tumors, drawing on data amassing to over 100,000 peptide sequences from these extended data sets. Moreover, several of these peptides showed in vitro immunogenicity across multiple melanoma patients. These observations highlight the breadth and complexity of the repertoire of immunogenic peptides that can be exploited therapeutically and suggest that spliced peptides are a major new class of tumor antigens.

Response to Comment on “A subset of HLA-I peptides are not genomically templated: Evidence for cis- and trans-spliced peptide ligands”

“…We believe that spliced peptides exist at a high prevalence, and we have recently shown their role in tumor antigen recognition (10).…”

Spliced HLA-bound peptides: a Black Swan event in immunology

“…Dalet et al [28] and Paes et al [24] showed that with increasing the size of the intervening sequence, the probability of both forward and reverse cis-splicing decreases, although without being wholly abolished. However, this trend was not observed in data-orientated approaches [12,13,23]. Two proposed mechanisms for peptide splicing are (i) direct transpeptidation [3,6] and (ii) carboxylic acid and amine condensation [6].…”

“…It has been suggested that peptide splicing is a stochastic process in the cell. However, we have recently found the repeated presentation of the same spliced peptides between biological replicates and even the same spliced peptides being presented by shared HLA alleles from different individuals [23]. It has also been shown in several in-vitro and in-cellulo experiments that both constitutive (standard) and immunoproteasomes can generate spliced peptides, with only slight differences in cleavage-site preferences [5,6,23].…”

“…However, we have recently found the repeated presentation of the same spliced peptides between biological replicates and even the same spliced peptides being presented by shared HLA alleles from different individuals [23]. It has also been shown in several in-vitro and in-cellulo experiments that both constitutive (standard) and immunoproteasomes can generate spliced peptides, with only slight differences in cleavage-site preferences [5,6,23]. This indicates that understanding the rules behind the generation of spliced peptides is essential to predict which spliced peptides are likely to be generated and presented from an antigen or restricted to a given HLA allotype.…”

“…This indicates that understanding the rules behind the generation of spliced peptides is essential to predict which spliced peptides are likely to be generated and presented from an antigen or restricted to a given HLA allotype. Two approaches are being used for finding the rules governing peptide splicing: (i) in-vitro PCPS [6,16,[24][25][26]] and (ii) data-orientated approaches that analyse the MS data from HLA-I-eluted spliced peptides [7,12,13,19,23].…”