Abstract Although neoantigen vaccines are proving effective in prolonging recurrence-free survival in patients with resected tumors, their impact is limited in cases of advanced metastatic disease. To improve patient outcomes, it is essential to understand the factors limiting vaccine efficacy. Here, we utilize the clinically relevant RNA-lipoplex vaccine platform, the MC-38 mouse tumor model, and single cell RNA sequencing (scRNAseq) to identify and investigate barriers limiting neoantigen vaccine activity. We find that vaccination generates polyepitotic CD8 T cells of multiple neoantigen specificities that deeply infiltrate tumor tissues. These neoantigen-specific CD8 tumor-infiltrating lymphocytes (TILs) exist along a diverse spectrum of effector T cell profiles, including those that have recently infiltrated, are interferon-stimulated, or are cytotoxic. Presence of these neoantigen-specific CD8 TILs is also associated with remodeling of the tumor microenvironment (TME) which is characterized by a shift of the myeloid compartment toward a more inflammatory and activated state, defined by expression of iNOS and CD80. Enhanced CD8 TIL infiltration and TME remodeling is collectively associated with complete regression of smaller, immature tumors, but only delayed the growth of larger, established tumors. Most interestingly, while vaccine-induced CD8 T cells are long-lived and functional in peripheral tissues, the abundance and transcriptional diversity of CD8 TILs is short-lived, rapidly declining following cessation of therapeutic neoantigen vaccination, coinciding with loss of tumor growth control. Following recent vaccination, CD8 TILs were broadly found to express a pro-apoptotic program, likely underlying their eventual decline. CD8 TILs that appeared to persist longer-term following this decline resemble TILs found in treatment-naive tumors and also express gene programs associated with stress and impaired activation. Importantly, recurrent vaccination replenishes cytotoxic and proliferative neoantigen-specific CD8 TILs and enhances anti-tumor activity. Collectively, these findings highlight the importance of optimizing therapeutic neoantigen vaccination schedules and combination therapies to promote the maintenance and function of vaccine-induced T cells in established tumors. Citation Format: Justin T. Gibson, Milena Hornburg, Sophie Lehar, Vincent Javinal, Lisa Liao, Mark J. McCarron, Tamaki N. Jones, Yoko Oei, Debra Dunlap, Kevin A. Marroquin, Amy A. Lo, Jan Bergmann, Ann-Jay Tong, Ariane Nissenbaum, Emily C. Freund, Aude-Hélène Capietto, Cecile C. de la Cruz, Ugur Sahin, Ira Mellman, Jill M. Schartner, Lélia Delamarre. Recurrent RNA-lipoplex vaccination replenishes neoantigen-specific CD8 tumor-infiltrating lymphocytes abstract. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr C068.
Gibson et al. (Wed,) studied this question.