Activity in vivo of in vitro expanded and adoptively transferred antigen-specific CD8+ T cells against neoepitopes that mediate tumor rejection 2306826

Abstract

A subset of somatic passenger mutations in cancer cells lead to generation of peptides which can be presented by MHC I molecules and recognized by CD8 T cells. Such peptides are tumor- specific and are termed neoepitopes. A small subset of neoepitopes can immunize mice against tumors in models of prophylaxis and some of them can be used to treat preexisting mouse tumors in models of therapy (see Srivastava J Clinical Investigation 2025 for review). We identified and selected antigen-specific CD8+ T cells using pMHC class I tetramers. A novel expansion method was applied to generate antigen-specific CD8+ T cells in vitro. Expanded antigen-specific T cells were adoptively transferred to tumor-bearing mice, and their antigen-specific clonal response, functional properties, and cell-fate decisions were assessed by flow cytometry following transfer. In addition, enriched whole tumor-infiltrating lymphocytes were examined with single-cell RNA-sequencing to characterize transcriptomic profiles. Here, we have analyzed antigen-specific CD8+ T cells against neoepitopes PDPRMUT, C3MUT and Ccdc65cMUT, all of which mediate tumor rejection of BALB/c Meth A fibrosarcoma (Ebrahimi-Nik et al. JCI Insight 2019, Nature Communications 2021, Singhaviranon et al. Nature Immunology 2025). We have developed methods of expansion in vitro of CD8+ T cells against these neoepitopes and have characterized their phenotypes in vitro, and after adoptive transfer into 10-day old Meth A bearing mice by multi-panel flow cytometry and RNASeq. These studies shed new light on the mechanisms by which neoepitope-specific CD8 T cells migrate to the tumor bed, proliferate, kill tumor cells, get exhausted and die. They also highlight the factors that limit the activity of neoepitope-specific CD8+ T cells in vivo and how these limitations can be addressed to obtain a better mechanistic understanding of neoepitope function. These mechanistic insights can also be used for a better immunotherapy of cancers. UCONN HEALTH Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)