Dataset Information

MHC-I peptidome analysis of human foreskin fibroblasts

ABSTRACT: Ribosome profiling (Ribo-seq) recently revealed the expression of thousands of short open reading frames (sORFs) in eukaryotic cells. They encode for a class of instable peptides, which evade experimental validation by whole-proteome mass spectrometry. Here, we show that computational elimination of experimental noise from Ribo-seq data unravels fundamental new aspects of sORF biology. Based on the revised annotation of cellular sORFs, we show that sORF-derived peptides are efficiently incorporated into MHC-I. sORFs thus encode a novel class of stress-responsive antigens in human cells. Our findings have broad implications on the functional, regulatory and immunogenic role of sORFs in fundamental cellular processes like infection and cancer.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Primary Cell

SUBMITTER: Florian Erhard

LAB HEAD: Stefan Stevanović

PROVIDER: PXD007203 | Pride | 2018-04-05

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	170204_MLu_HF99-7_Mock_W_20_DDA1_400-650mz_msms7.raw	Raw
	170204_MLu_HF99-7_Mock_W_20_DDA2_400-650mz_msms8.raw	Raw
	170204_MLu_HF99-7_Mock_W_20_DDA3_400-650mz_msms9.raw	Raw
	170204_MLu_HF99-7_infiziert_W_20_DDA1_400-650mz_msms10.raw	Raw
	170204_MLu_HF99-7_infiziert_W_20_DDA2_400-650mz_msms11.raw	Raw

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Publications

Improved Ribo-seq enables identification of cryptic translation events.

Erhard Florian F Halenius Anne A Zimmermann Cosima C L'Hernault Anne A Kowalewski Daniel J DJ Weekes Michael P MP Stevanovic Stefan S Zimmer Ralf R Dölken Lars L

Nature methods 20180312 5

Ribosome profiling has been used to predict thousands of short open reading frames (sORFs) in eukaryotic cells, but it suffers from substantial levels of noise. PRICE (https://github.com/erhard-lab/price) is a computational method that models experimental noise to enable researchers to accurately resolve overlapping sORFs and noncanonical translation initiation. We experimentally validated translation using major histocompatibility complex class I (MHC I) peptidomics and observed that sORF-deriv ...[more]

PMID: 29529017

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Project description:Infection or reactivation with human cytomegalovirus (HCMV) is still a major cause of morbidity and mortality in patients undergoing solid organ transplantation or allogeneic stem cell transplantation (alloSCT). Recent studies have shown that protection against latent HCMV infection relies on a much broader antigen spectrum than previously expected. The identification of novel immunogenic HCMV-derived peptides that facilitate HCMV control is therefore essential to improve immune monitoring of HCMV-specific T cells and HCMV vaccine development. In particular, only a few HCMV-derived peptides have been described for less common HLA alleles, such as HLA-A*03:01 and HLA-B*15:01, limiting the implementation of these techniques for a substantial number of patients. Here, we identified novel HCMV-derived HLA-A*03:01- and HLA-B*15:01-restricted immunogenic peptides by an innovative combined in vitro and in silico approach. We utilized the computational tools Peptide-PRISM (1) and PRICE (2) to analyze mass spectrometric (MS) and ribosome sequencing (ribo-seq) datasets, respectively. To refine our in silico approach, we utilized machine learning to rate the identified peptide candidates based on their translational activity, binding affinity and positioning within the small open reading frames (sORFs), with the goal of assessing their likelihood to be presented on HLA-A*03:01 and HLA-B*15:01 molecules. After identifying the highest-scoring 49 canonical and 49 cryptic potentially immunogenic peptides for each HLA allele, we assessed their immunogenicity by screening HCMV-seropositive and -seronegative healthy donors, as well as alloSCT patients for their peptide-specific T-cell responses. We used a stimulation procedure in two consecutive steps: peripheral blood cells (PBMCs) were stimulated starting with peptide pools and subsequently with potentially reactive single peptides. In vitro T-cell stimulation resulted in the direct identification of three canonical and one cryptic HLA-A*03-restricted immunogenic peptides as well as five canonical and one cryptic HLA-B*15-restricted immunogenic peptide, with a specific IFNγ+/CD8+ T cell response of ≥ 0.02%. Highest T-cell responses were identified against two HLA-A*03-restricted and three HLA-B*15-restricted canonical peptides with up to 3.42% of IFNγ+/CD8+ T cells in patients 180 days post alloSCT. In conclusion, we identified specific T-cell responses against eight canonical and two cryptic HLA-A*03:01- and HLA-B*15:01-restricted peptides in healthy individuals and post-transplanted patients and which were classified as immunogenic. Of these, six peptides are novel HCMV-derived, immunogenic peptides according to the UniProt database, broadening the spectrum of immunogenic HCMV-derived peptides for personalized immune monitoring and vaccine development.