Soluble HLA peptidome of pleural effusions is a valuable source for tumor antigens

Abstract

Background: Soluble human leucocyte antigen (sHLA) molecules, released into the plasma, carry their original peptide cargo and provide insight into the protein synthesis and degradation schemes of their source cells and tissues. Other body fluids, such as pleural effusions, may also contain sHLA-peptide complexes, and can potentially serve as a source of tumor antigens since these fluids are drained from the tumor microenvironment. We explored this possibility by developing a methodology for purifying and analyzing large pleural effusion sHLA class I peptidomes of patients with malignancies or benign diseases.

Methods: Cleared pleural fluids, cell pellets present in the pleural effusions, and the primary tumor cells cultured from cancer patients' effusions, were used for immunoaffinity purification of the HLA molecules. The recovered HLA peptides were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and the resulting LC-MS/MS data were analyzed with the MaxQuant software tool. Selected tumor antigen peptides were tested for their immunogenicity potential with donor peripheral blood mononuclear cells (PBMCs) in an in vitro assay.

Results: Mass spectrometry analysis of the pleural effusions revealed 39,669 peptides attributable to 11,305 source proteins. The majority of peptides identified from the pleural effusions were defined as HLA ligands that fit the patients' HLA consensus sequence motifs. The membranal and soluble HLA peptidomes of each individual patient correlated to each other. Additionally, soluble HLA peptidomes from the same patient, obtained at different visits to the clinic, were highly similar. Compared with benign effusions, the soluble HLA peptidomes of malignant pleural effusions were larger and included HLA peptides derived from known tumor-associated antigens, including cancer/testis antigens, lung-related proteins, and vascular endothelial growth factor pathway proteins. Selected tumor-associated antigens that were identified by the immunopeptidomics were able to successfully prime CD8⁺ T cells.

Conclusions: Pleural effusions contain sHLA-peptide complexes, and the pleural effusion HLA peptidome of patients with malignant tumors can serve as a rich source of biomarkers for tumor diagnosis and potential candidates for personalized immunotherapy.

Keywords: antigen presentation; antigens; immunotherapy; lung neoplasms; tumor biomarkers.

Figure 1

Experimental design. Pleural effusions were collected from cancer and non-cancer patients, cells were separated and cultures were grown, when possible. HLA-peptide complexes were immunoaffinity-purified, peptides were separeted and analyzed by mass spectrometer. Peptides were considered HLA ligands after filtration of known contaminants, and if they were 8–14 amino-acids-long and matched the HLA allele consensus motifs of each patient, based on their tissue typing and online analysis with the NetMHCpan V.4.1 server http://www.cbs.dtu.dk/services/NetMHCpan/. Tumor antigens were identified from the peptides lists and candidates were tested for an immunogenic response. The figure was created using BioRender (https://biorender.com/). HLA, human leucocyte antigen; LC-MS/MS, liquid chromatography and tandem mass spectrometry.

Figure 2

Optimization of pleural effusion volumes for HLA peptidome purification. (A) Primary tumor cells cultured from Patient 3 pleural effusion samples immunostained for TTF1 (green) and EpCAM (red). Nuclei were stained with DAPI (blue). (B) Western blot analysis of 10% of the protein fraction of 10–400 mL pleural effusion HLA purifications from patient 3, stained with anti-pan-HLA antibody. (C) Venn diagram of peptides identified from HLA purification of 10 (light blue), 40 (orange), 100 (green) and 400 (pink) mL of the pleural effusions collected from patient 3. The scaling represents total number of identified peptides. The number of putative HLA ligands and total number of identified peptides appear on the graph. The diagram was produced using the R package VennEuler (V.1.1–0). (D) Profile plot of log₂ liquid chromatography-mass spectrometry (LC-MS) intensities of peptides identified from different volumes of pleural effusions. The gray lines indicate the log₂ intensities of the identified peptides and the colored lines are selected peptides. EpCAM, epithelial cell adhesion molecule; HLA, human leucocyte antigen; TTF1, thyroid transcription factor 1.

Figure 3

Characterization of the identified HLA peptidome. (A) Length distributions of the peptides identified in the pleural fluids (left) and bulk cells (right) of all patients. The different colors of the circles represent different patients. The percentages were calculated for each length before data filtration. (B) Distribution of the HLA peptides to their presenting allotypes. The gray color represents the peptides that are either unassigned to a specific HLA allele by NetMHCpan V.4.1, are known contaminants in MaxQuant, and/or are longer than 14 amino acids. The colorful bars indicate the number of ligands attributed to HLA-A, HLA-B or HLA-C, in orange, blue or green, respectively, according to NetMHCpan V.4.1. (C) Scatter plots of log₂ liquid chromatography-mass spectrometry (LC-MS) intensities of peptides identified from different pleural effusions samples of Patients 11 (left) and 13 (right). The dot colors indicate the peptides length. Pearson correlation value is indicated in each graph. HLA, human leucocyte antigen.

Figure 4

The intensities of the peptides identified in the pleural effusion, bulk cells, cultured cells, and plasma of Patient 13, are correlated to each other. Scatter plots of the log₂ LC-MS intensities of the peptides, identified from different sources, are shown. The colors represent the peptide length. Venn diagrams near each plot indicate the number of peptides identified in each sample. Arrows point to the intensities of the peptide MPYPSKSNQEVL, originating from anaplastic lymphoma kinase (ALK). Pearson correlations are indicated in each graph. LC-MS, liquid chromatography and mass spectrometry.

Figure 5

The number of peptides per HLA allotype, presented on the membranal HLA and the soluble HLA, are correlated to each other. The number of peptides associated with specific HLA allotype, from malignant and benign pleural effusions, is represented by circles and squares, respectively. Some of the allotypes are annotated (see online supplemental table S10 for the complete list). HLA, human leucocyte antigen.

Figure 6

peHLA peptidome originate from tumor-associated genes and includes immunogenic peptides. (A) 4-1BB expression was assayed by FACS analysis of purified naïve CD8⁺ T cells, from healthy HLA-B*35:02 positive donor, following co-culturing with peptide-loaded autologous DCs. Pseudo-color dot plots display the responses for the five HLA peptides and the negative control (cells stimulated with empty DCs). upper numbers in the quadrants show percentages of CD8⁺/4-1BB⁺ cells. (B) The media from restimulated T cells was collected and assessed for the concentration of IFNγ by ELISA. P values were determined by one-way ANOVA test relative to the negative control. n=3, data are represented as mean±SEM *p<0.05, ****p<0.0001. (C) Examples of tumor antigens which are the source of many HLA peptides, including tumor antigens that were identified in the KEGG pathways in cancer database. The antigens are presented with the number of identified peHLA peptides derived from each source protein in each patient (see online supplemental table S9 for the complete list). ANOVA, analysis of variance; DC, dendritic cell; HLA, human leucocyte antigen; peHLA, pleural effusion soluble HLA.