Gating strategy to identify cytokine-secreting cells by cytokine capture assay. The acquired cells were gated based on their size and granularity as lymphocytes excluding cell debris. Then we gated on single cells to remove cell duplexes and clusters. Apoptotic cells were gated out next by gating on pacific orange (PO)-negative cells (live cells). The respective T-cell (TC) population was identified by surface staining with anti-CD4 PerCP-Cy5.5, and anti-CD8-V450 antibodies. The cytokine-positive cell gate was placed according to a FMO control (exemplarily shown for IFN-�) and the frequency of cytokine-secreted cells in samples stimulated with IgG1 peptide (negative control) or the antigen of interest (exemplarily shown for CFL1) were determined based on that gate.
ARTICLE ABSTRACT
Purpose: Successful immunotherapies for IDHmut gliomas require better knowledge of T-cell target antigens. Here, we elucidated their antigen repertoire recognized by spontaneous T-cell responses using an unbiased proteomic approach.Experimental Design: Protein fractionations of tissue lysates from IDHmut gliomas (n = 4) were performed. Fractions were tested by IFNγ ELISpot assay for recognition through patients' T cells. Proteins of immunogenic fractions were identified by mass spectrometry and validated by in silico-predicted synthetic long peptides in patients of origin, additional IDHmut glioma patients (n = 16), and healthy donors (n = 13). mRNA and protein expression of immunogenic antigens was analyzed in tumor tissues and IDHmut glioma stem-like cells (GSC). HLA-A*02–restricted T-cell epitopes were functionally determined by short peptides and numbers of antigen-specific T cells by HLA-peptide tetramer analysis.Results: A total of 2,897 proteins were identified in immunogenic tumor fractions. Based on a thorough filter process, 79 proteins were selected as potential T-cell antigens. Twenty-six of these were recognized by the patients’ T cells, and five of them (CRKII, CFL1, CNTN1, NME2, and TKT) in up to 56% unrelated IDHmut glioma patients. Most immunogenic tumor-associated antigens (TAA) were expressed in IDHmut gliomas and GSCs, while being almost absent in normal brain tissues. Finally, we identified HLA-A*02–restricted epitopes for CRKII, NME2, and TKT that were recognized by up to 2.82% of antigen-specific peripheral cytotoxic T cells in IDHmut glioma patients.Conclusions: By analyzing the repertoire of T-cell target antigens in IDHmut glioma patients, we identified five novel immunogenic TAAs and confirmed their expression on IDHmut tumors and GSCs. Clin Cancer Res; 24(12); 2951–62. ©2018 AACR.
