An experimental method to delineate responses from pre-existing memory and induced naive T cells in neoepitope candidate screening

Abstract

Abstract Identifying tumor-specific T cells is critical to the development of T-cell based immunotherapies. As a result, computational tools have been developed to predict the neoepitopes present within a tumor. A common experimental approach used to screen neoepitope candidates is the long-term in vitroexpansion of patient peripheral blood mononuclear cells (PBMC) with candidate peptides-followed by a fluorospot to detect antigen-specific responses. Although this approach allows for high-throughput screening of candidates, it cannot distinguish between responses from pre-existing memory cells versus from naïve cells that were primed in vitro. We have developed an experimental method to fulfill this need. We designed a negative control peptide pool of 15-mers with no sequential similarity to any known antigens. Through the use of this pool with depletion of naïve T cells from PBMC, we show in 12 healthy donors that this megapool successfully quantifies signal that is a result of naïve cell proliferation. Importantly, we applied this approach to screen pools of neoepitopes in this cohort of donors. We found that the majority of the neoepitope-specific responses detected were derived from induced naïve cells whereas only a maximum of 3 donors had pre-existing memory to any given neoepitope pool. This work underscores the applicability and relevance of our method in characterizing fluorospot signals and provides the foundation for others to study the role of these various T cell responses in therapeutic outcomes. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship Program under Grant no. 2038238. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.