Abstract
Neoepitope-specific T-cell responses have been shown to induce durable clinical responses in patients with advanced cancers. We explored the recognition patterns of tumor-infiltrating T lymphocytes (TILs) from patients with glioblastoma multiforme (GBM), the most fatal form of tumors of the central nervous system. Whole-genome sequencing was used for generating DNA sequences representing the entire spectrum of ‘private’ somatic mutations in GBM tumors from five patients, followed by 15-mer peptide prediction and subsequent peptide synthesis. For each mutated peptide sequence, the wildtype sequence was also synthesized and individually co-cultured with autologous GBM TILs, which had been expanded in vitro with a combination of interleukin (IL)-2, IL-15 and IL-21. After seven days of culture, interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α) and/or IL-17A production was measured by ELISA in culture supernatants, and used as an epitope-specific immune response readout. Mutated peptides that induced a strong cytokine response were considered to contain legitimate neoepitopes. TILs from 5/5 patients with GBM exhibited specific immune reactivity profiles to the nominal target peptides, defined by IFN-γ and/or TNF-α production, as well as IL-17A. Neoepitopes, defined by mutated peptides inducing IFN-γ and/or TNF-α production without or only minimal reactivity to the wildtype sequences, were found for each individual patient. CD8+ TILs dominated the patients’ responses to private neoepitopes. The present study shows that neoepitope-specific TIL reactivity constitutes an important arm of anti-tumor immune responses in patients with GBM, and thus a powerful tool for developing next-generation personalized immunotherapies.
Highlights
The identification of T-cell epitopes provided from host-derived targets is crucial in developing personalized treatments for patients with advanced cancer
A heat map depicting cytokine responses (IFN-γ, tumor necrosis factor (TNF-α) and/or IL-17A) following a 7-day in vitro stimulation assay with the wildtype as well as mutated peptide sequences was generated for each patient (Figure 2)
The present study describes T-cell epitope mapping that combines whole-exome sequencing, identification of private mutations in a patient’s tumor DNA, synthesis of the respective mutated peptide sequence as well as the corresponding wildtype sequence for comparison and testing T-cell responses directed against specific mutated and wildtype epitopes found within the peptide sequences
Summary
The identification of T-cell epitopes provided from host-derived targets is crucial in developing personalized treatments for patients with advanced cancer. Epitopes are identified based on the amino acid sequence derived from antigens recognized by a specific population of T cells defined by immune reactivity, i.e. cytokine production, usually measured as antigen-induced interferon gamma (IFN-γ) responses This process does allow to www.oncotarget.com identify new cancer-specific targets, and to identify T-cell receptors (TCR) that can be used in the development of immunotherapies. Screening of cDNA libraries encoding tumor-associated antigens (TAAs) has been used extensively; this approach is labor-intensive and minimally effective, as larger transcripts and weakly expressed transcripts cannot be cloned This method eliminates identification of some mutated antigens since it does not cover post-transcriptional modifications of protein antigens [2]. In the TMG setting, only a small portion of the gene around the mutation is synthesized, and its reaction with autologous T cells is tested to identify whether the predicted neoepitopes are naturally processed and presented to the immune system [6] based on the assumption that the surrogate antigenpresenting cells process and present the neoepitopes in a similar fashion to tumor cells
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