Dataset Information

Immunopeptidomic profiling of HLA-A2-positive triple negative breast cancer

ABSTRACT: Here we analysed the immunopeptidomes of 6 HLA-A2-positive triple negative breast cancer (TNBC) samples by nano-ultra performance liquid chromatography tandem mass spectrometry (nUPLC-MS2).

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Breast

DISEASE(S): Triple-receptor Negative Breast Cancer

SUBMITTER: Nicola Ternette

LAB HEAD: Dr Nicola Maria Nathalie Ternette

PROVIDER: PXD009738 | Pride | 2018-06-29

REPOSITORIES: Pride

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Publications

Immunopeptidomic Profiling of HLA-A2-Positive Triple Negative Breast Cancer Identifies Potential Immunotherapy Target Antigens.

Ternette Nicola N Olde Nordkamp Marloes J M MJM Müller Julius J Anderson Amanda P AP Nicastri Annalisa A Hill Adrian V S AVS Kessler Benedikt M BM Li Demin D

Proteomics 20180601 12

The recent development in immune checkpoint inhibitors and chimeric antigen receptor (CAR) T-cells in the treatment of cancer has not only demonstrated the potency of utilizing T-cell reactivity for cancer therapy, but has also highlighted the need for developing new approaches to discover targets suitable for such novel therapeutics. Here we analyzed the immunopeptidomes of six HLA-A2-positive triple negative breast cancer (TNBC) samples by nano-ultra performance liquid chromatography tandem ma ...[more]

PMID: 29786170

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Project description:Human leukocyte antigen (HLA) - B*51:01 and endoplasmic reticulum aminopeptidase 1 (ERAP1) are two strong predisposing genetic factors to Behçet's disease (BD). Previous studies have focused on two subgroups of HLA-B*51 peptidome containing Proline (Pro) or Alanine (Ala) at position 2 (P2). Little is known about the unconventional non-Pro/Ala2 HLA-B*51-bound peptides. We aimed to study the features of this novel sub-peptidome, and investigate its regulation by ERAP1. CRISPR-Cas9 was used to generate an HLA-ABC-knockout HeLa cell line (HeLa.ABC-KO), which was subsequently transduced to express HLA-B*51:01 (HeLa.ABC-KO.B51). ERAP1 was silenced using lentiviral shRNA. Peptides bound to HLA-B*51:01 were eluted and analyzed by Mass Spectrometry. The characteristics of non-Pro/Ala2, Pro2 and Ala2 peptides, and their alteration by ERAP1 silencing were investigated. Effects of ERAP1 silencing on cell surface expression of HLA-B*51:01 were studied using flow cytometry. More than 20% of peptides eluted from HLA-B*51:01 lacked Proline or Alanine at P2. This unconventional group of HLA-B*51:01-bound peptides was enriched for 8-mers (with fewer 9-mers) compared to Pro2 and Ala2 sub-peptidomes, and had similar N-terminal and C-terminal residue usages to Ala2 peptides with the exception of the less abundant Leucine at position Ω. Knockdown of ERAP1 increased the percentage of non-Pro/Ala2 to approximately 40%, increased the percentage of longer (10-mer and 11-mer) peptides eluted from HLA-B*51:01 complexes, and abrogated the predominance of Leucine at P1. Interestingly knockdown of ERAP1 altered the length and N-terminal residue usage of non-Ala2&Pro2 and Ala2 but not the Pro2 peptides. Finally, ERAP1 silencing regulated the expression levels of cell surface HLA-B*51 in a cell type - dependent manner. The HLA-B*51:01 peptidome includes a surprisingly high proportion of unconventional non-Pro/Ala2 peptides which are increased by ERAP1 silencing, mimicking the loss-of-function BD risk variant.

Project description:Comparison of gene expression from subjects who resolved or formed pustules to H.ducreyi. In human inoculation experiments, the cutaneous immune response to Haemophilus ducreyi consists of serum, PMN, macrophages, T cells and myeloid DC. In reinfection experiments, some subjects form pustules twice (PP group) or resolve infection twice (RR group). Although pustule formation is associated with serum resistance and phagocytic failure, there are no differences in the ability of isolated phagocytes or serum obtained from PP and RR subjects to ingest or kill H. ducreyi. To identify the basis for differential host susceptibility to H. ducreyi, we used microarrays to profile gene expression in infected and uninfected tissue and monocyte-derived DC obtained from PP and RR subjects. In infected tissue, both groups had a core response to H. ducreyi. Many additional transcripts that signify active immune function were upregulated exclusively in RR tissue, while PP tissue exclusively contained differentially regulated transcripts consistent with immune dysregulation. The core response of infected DC from both groups was typical of a DC1 response. RR DC exclusively expressed many additional transcripts indicative of DC1 function, while PP DC uniquely expressed differentially regulated transcripts characteristic of both DC1 and DCreg. The data suggest that DC from PP and RR subjects are prewired to respond differentially to H. ducreyi. Experiment Overall Design: Six healthy adult volunteers (5 females, 1 male, 36 Â± 13 yrs, mean age Â± SD) who had been infected with H. ducreyi twice were inoculated a third time. Each volunteer was inoculated at 3 sites on the upper arm with live H. ducreyi 35000HP (a human passaged isolate of strain 35000) and at 1 site with sterile PBS. Forty-eight hours after inoculation, lesion size was measured and RNA was isolated from the infected site that had the largest diameter. RNA was also obtained from the PBS control site (uninfected site). We obtained peripheral blood 6 to 12 months after inoculation of the 6th subject and derived myeloid DC in vitro. In brief, CD14+ peripheral blood monocytes were isolated from PBMC by positive selection magnetic beads (Miltenyi Biotec, Auburn, CA) and grown in the presence of recombinant human (rh) IL-4 (1 ng/ml) and rhGM-CSF (0.2 ng/ml) (R&D Systems, Minneapolis, MN). The cells were HLA-DR+, CD86+ CD40+, CD3-, CD14-, and CD19- by flow cytometry. The experiment was done in pairs so that DC from 1 RR subject were exposed to the same inoculum as DC from 1 PP subject. DC were incubated with nonopsonized H. ducreyi for 90 minutes at an MOI of 30:1. The DC were washed to remove non-associated bacteria, and incubated an additional 22.5 hours. Cells were collected and used for microarray analysis. Supernatants were collected and analyzed for cytokines using the Human Th1/Th2 II Cytometric Bead Array Kit per manufacturerâs instructions (BD Biosciences).

Project description:Virus resistances that are recessively inherited are associated with loss-of-susceptibility resistance alleles. Resistance to Watermelon mosaic virus (WMV) of melon accession TGR-1551 is expressed as a drastic reduction of the virus titer, and is recessively inherited. In this work, viral RNA accumulation was measured in TGR-1551 and in susceptible WMV-infected melon plants by real time quantitative PCR (qPCR), and gene expression of 17,443 unigenes represented in a melon microarray was monitored in a time-course experiment. Virus accumulation was higher in inoculated cotyledons of the resistant genotype up to 7 days post-inoculation; from this time on, virus accumulation was much higher in plants of the susceptible genotype. Microarray experiments were carried with samples from inoculated cotyledons at 1 and 3 dpi to monitor early changes in response to virus infection, and at 7 dpi. Samples from systemically infected leaves harvested at 15 dpi were also included in the analysis. Results showed much more profound transcriptomic alterations in resistant plants compared to susceptible ones. Analyses of gene expression profiles reveal deep and extensive transcriptomic alterations in TGR-1551 plants, many of them involving pathogen response-related genes. Overall, data suggested that resistance to WMV in TGR-1551 is associated with a defense response, contrasting with its recessive nature. Two melon genotypes have been used to analyse transcriptomic responses to infection by Watermelon mosaic virus: Tendral (susceptibel to WMV) and TGR-1551 (resistant to WMV). For each genotype, 60 melon seedlings were inoculated with WMV-M116 and another 60 were mock-inoculated. Cotyledons of 10 plants were harvested at 1, 3, 5, 7, 9 and 15 dpi. At 15 dpi, the systemically infected second true leaf was also harvested. To reduce variability, each biological replicate used in this study was prepared by mixing the RNA extracts from 2 or 4 mock or WMV-inoculated cotyledons, respectively, or from 3 melon leaves. Samples (WMV infected and mock inoculated) corresponding to cotyledons at 1, 3 and 7 dpi, and leaves at 15 dpi were used for microarray hybridisations, three biological replicates for each one, leading to a total of 48 samples.

Dataset Information

Immunopeptidomic profiling of HLA-A2-positive triple negative breast cancer

Publications

Immunopeptidomic Profiling of HLA-A2-Positive Triple Negative Breast Cancer Identifies Potential Immunotherapy Target Antigens.

Similar Datasets

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