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. 2018 Mar;17(3):533-548.
doi: 10.1074/mcp.TIR117.000383. Epub 2017 Dec 14.

High-throughput and Sensitive Immunopeptidomics Platform Reveals Profound Interferonγ-Mediated Remodeling of the Human Leukocyte Antigen (HLA) Ligandome

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Free PMC article

High-throughput and Sensitive Immunopeptidomics Platform Reveals Profound Interferonγ-Mediated Remodeling of the Human Leukocyte Antigen (HLA) Ligandome

Chloe Chong et al. Mol Cell Proteomics. .
Free PMC article

Abstract

Comprehensive knowledge of the human leukocyte antigen (HLA) class-I and class-II peptides presented to T-cells is crucial for designing innovative therapeutics against cancer and other diseases. However methodologies for their purification for mass-spectrometry analysis have been a major limitation. We designed a novel high-throughput, reproducible and sensitive method for sequential immuno-affinity purification of HLA-I and -II peptides from up to 96 samples in a plate format, suitable for both cell lines and tissues. Our methodology drastically reduces sample-handling and can be completed within five hours. We challenged our methodology by extracting HLA peptides from multiple replicates of tissues (n = 7) and cell lines (n = 21, 108 cells per replicate), which resulted in unprecedented depth, sensitivity and high reproducibility (Pearson correlations up to 0.98 and 0.97 for HLA-I and HLA-II). Because of the method's achieved sensitivity, even single measurements of peptides purified from 107 B-cells resulted in the identification of more than 1700 HLA-I and 2200 HLA-II peptides. We demonstrate the feasibility of performing drug-screening by using ovarian cancer cells treated with interferon gamma (IFNγ). Our analysis revealed an augmented presentation of chymotryptic-like and longer ligands associated with IFNγ induced changes of the antigen processing and presentation machinery. This straightforward method is applicable for basic and clinical applications.

Figures

Fig. 1.
Fig. 1.
Outline of the high-throughput immunopurification workflow using a plate format. A, Tissues are first homogenized, lysed with mild detergents and cleared with a centrifugation step. B, To enable sequential loading of the lysates on multiple affinity resins, cleared lysates are loaded on stacked plates containing firstly, Pro-A beads for depletion of tissue endogenous antibodies, then anti-HLA class I and II antibodies cross-linked to Pro-A beads for direct enrichment of HLA class I and II complexes. C, Affinity plates containing the captured HLA complexes are separated, washed individually and stacked on C18 plates. HLA class I and II complexes are then eluted on the C18 plates. Peptide and protein fractions are then recovered separately. Each step is timed with the hourglass symbol that is equivalent to about one hour.
Fig. 2.
Fig. 2.
In-depth and sensitive analysis of HLA-Ip and HLA-IIp at 1% FDR for peptide identifications. A, Number of unique HLA-Ip (blue bars) and (B) HLA-IIp (green bars) identified for B- and T-cell lines and individual tissue samples, and in total (gray bars). C, Length distribution of HLA-Ip and (d) of HLA-IIp. D, Average number of HLA-Ip (blue bars) and (E) HLA-IIp (green bars) identified in triplicates in lysate volumes equivalent to 10, 30, 50, 70 and 100 -million CD165 cells. Data is represented as mean ± S.D. F, Distribution of intensities of HLA-Ip and (G) HLA-IIp detected in the samples of 100 million cells and those detected in samples of both 10 million and 100 million cells.
Fig. 3.
Fig. 3.
Assessment of intra-plate reproducibility. A, Overlap in the frequency of HLA-Ip and (B) HLA-IIp identified in three plate replicates of RA957 samples. C, Intra-plate reproducibility calculated by Pearson correlations of log2 transformed intensities of HLA-Ip and (D) HLA-IIp identified across the different MS measurements. E, Examples of comparative semi-quantitative analysis of HLA-Ip detected in two MS measurements (referred here as technical MS replicates) of one RA957 sample and (F) of two representative plate replicates of RA957 samples. Values of the Pearson correlation are indicated.
Fig. 4.
Fig. 4.
Label-free semi-quantitative comparative analysis of IFNγ modulated immunopeptidome. A, Reproducibility calculated by Pearson correlations of log2 transformed intensities from HLA-Ip of control and IFNγ -treated samples across the different MS measurements. B, Number of HLA-Ip identified from UWB.1 289 ovarian cancer cells untreated (control) and treated with IFNγ. The number of peptides identified with (gray) and without (blue) matching identifications across the treated and untreated samples and the average values of the Pearson correlations are indicated. C, Summed peptide intensities identified in each of the IFNγ treated and control samples. D, Volcano plot summarizing unpaired t test analysis of the immunopeptidome of IFNγ treated versus untreated cells. Peptides located above the lines are statistically significantly modulated in their level of presentation (FDR = 0.01, S0 = 1). All peptides derived from proteins related to immunity are highlighted in pink. Selected up-regulated peptides were highlighted in red, corresponding to well known intracellular mediators of IFNγ signaling. E, Volcano plot of unpaired t test analysis of the proteome of IFNγ treated versus untreated cells. Proteins located above the lines are statistically significantly modulated in their expression level (FDR = 0.01, S0 = 0.2). Selected proteins involved were similarly highlighted.
Fig. 5.
Fig. 5.
Impact of IFNγ on global features of HLA class-I repertoire. A, Peptides were assigned to the different HLA allotypes based on binding affinity predictions and their binding motifs depicted with sequence logos. B–C, Volcano plots summarizing unpaired t test analysis of the immunopeptidome of IFNγ treated versus untreated cells. Peptides located above the lines are statistically significantly modulated in their level of presentation (FDR = 0.01, S0 = 1). All chymotryptic-like (B) and tryptic-like ligands (C) were highlighted, respectively. D–E, Length distribution of peptides uniquely identified in IFNγ treated (orange) or control (blue) samples according to their chymotryptic- (D) or tryptic-like (E) properties. F–G, Intensity changes on IFNγ treatment were calculated for longer peptides against their shorter versions for both C- or N-terminal extensions: Normalized log2-intensity difference = log2((IFNγlong-ctrllong)/(IFNγshort-ctrlshort)). (H) C-terminal nested versions were grouped based on whether their extended peptides remained tryptic-like (T→T), chymo-tryptic like (C→C), or if their specificities were switched (C→T or T→C). Log2-intensity changes on IFNγ treatment were calculated for longer peptides against their shorter versions (I) For T→C and C→C peptide pairs, the sequence logos around the cleavage site of the long peptides (C-terminal P1–5, downstream P'1–5) are depicted (one-sided t test, p value * < 0.1; ** < 0.05; and *** < 0.01).

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