Abstract
All nucleated mammalian cells express major histocompatibility complex (MHC) proteins that present peptides on cell surfaces for immune surveillance. These MHC-presented peptides (pMHC) are necessary for directing T-cell responses against cells harboring non-self antigens derived from pathogens or from somatic mutations. Alterations in tumor-specific antigen repertoires — particularly novel MHC presentation of mutation-bearing peptides (neoantigens) — can be potent targets of anti-tumor immune responses. Here we employed an integrated genomic and proteomic antigen discovery strategy aimed at measuring how interferon gamma (IFN-γ) alters antigen presentation, using a human lymphoma cell line, GRANTA-519. IFN-γ treatment resulted in 126 differentially expressed proteins (2% of all quantified proteins), which included components of antigen presentation machinery and interferon signaling pathways, and MHC molecules themselves. In addition, several proteasome subunits were found to be modulated, consistent with previous reports of immunoproteasome induction by IFN-γ exposure. This finding suggests that a modest proteomic response to IFN-γ could create larger alteration to cells’ antigen/epitope repertoires. Accordingly, MHC immunoprecipitation followed by mass spectrometric analysis of eluted peptide repertoires revealed exclusive signatures of IFN-γ induction, with 951 unique peptides reproducibly presented by MHC-I and 582 presented by MHC-II. Furthermore, an additional set of pMHCs including several candidate neoantigens, distinguished control and the IFN-γ samples by their altered relative abundances. Accordingly, we developed a classification system to distinguish peptides which are differentially presented due to altered expression from novel peptides resulting from changes in antigen processing. Taken together, these data demonstrate that IFN-γ can re-shape antigen repertoires by identity and by abundance. Extending this approach to models with greater clinical relevance could help develop strategies by which immunopeptide repertoires are intentionally reshaped to improve endogenous or vaccine-induced anti-tumor immune responses and potentially anti-viral immune responses.
Highlights
Peptides presented on the cell surface by major histocompatibility complex proteins (MHC; known as human leukocyte antigen (HLA) in humans) are a fundamental component of immunological diseases, including infection, autoimmunity, allergy, and cancer
To better understand how IFN-g alters pMHC repertoires, we developed a categorization scheme which distinguishes pMHC resulting from differential expression versus from differential presentation
We showed that combining transcriptomics, proteomics, and immunopeptidomics can be a powerful strategy for uncovering treatment-specific effects on antigen presentation
Summary
Peptides presented on the cell surface by major histocompatibility complex proteins (MHC; known as human leukocyte antigen (HLA) in humans) are a fundamental component of immunological diseases, including infection, autoimmunity, allergy, and cancer. Decades of seminal research established robust mechanisms for antigen presentation by MHC-I and MHC-II molecules [1,2,3,4] Due to their importance across the span of human disease, intense efforts have built pMHC prediction algorithms to suggest which of trillions of possible peptides could have therapeutic utility [5]. Empirical data generated by high-throughput mass spectrometry methods have increasingly suggested large numbers of naturally processed peptide ligands do not fit conventional models [6, 7] Potential reasons behind these discrepancies include altered cell states, proteasomal changes, post-translational modifications, and modulation by antigen presentation-associated aminopeptidases, which these models largely cannot consider. Modeling cell state-dependent changes in antigen presentation machinery is difficult, but can be readily assessed by routine proteomic surveys
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