Ex vivo pre-activation shifts the in vivo differentiation of adoptively transferred CD8+ T cells in a melanoma model

Adoptive transfer of TCR-specific CD8⁺ T cells represents a powerful experimental platform for investigating tumor-specific CD8⁺ T cell responses within the framework of anti-tumor immunity. Genetic modulation of these transferred cells provides a robust strategy to elucidate the intrinsic molecular mechanisms underlying T cell differentiation and functionality, thereby offering critical insights to optimize tumor-specific CD8⁺ T cell antitumor immunity in cancer immunotherapy. A key aspect of this approach is the ex vivo activation of primary T cells, which raises important questions regarding the impact of pre-activation on subsequent T cell differentiation. In this study, we explored the differentiation trajectories of pre-activated CD8⁺ T cells and performed a comprehensive characterization of their epigenetic and transcriptional profiles using a murine melanoma model. Our findings revealed that ex vivo pre-activation not only attenuates progression towards terminal exhaustion in the tumor-draining lymph nodes (TdLNs) but also enhances the stem-like characteristics of CD8⁺ T cells within the tumor microenvironment (TME). Leveraging comprehensive ATAC-seq and RNA-seq analyses, we demonstrated that pre-activation modulates the epigenetic landscape and transcriptional profile of CD8⁺ T cells, fostering effector-related differentiation in the TdLNs while promoting stemness-associated programming in the TME. These findings highlight the profound influence of ex vivo pre-activation on the differentiation pathways of tumor-specific CD8⁺ T cells, underscoring the necessity of taking these experimental framework-induced discrepancies into consideration for more accurate data interpretation in relevant researches.