Background Fate decision in CD8⁺ T cells refers to the process of differentiation that occurs when a naïve T cell enters into effector, memory, exhausted, or other states. Multiple models of fate decision have been proposed, but the exact determinants of T cell fate are unknown. Evidence in LCMV and cancer models support the notion that signals during T cell priming are especially important in deciding T cell fate and that fate decisions are made early in the priming process. Priming interactions are highly complex, requiring the integration of multiple signals. Each of these signals can vary in strength and it is the sum of these signals that decides the T cell response. Thus, it is unsurprising that we currently do not know the combinatorial specificities and molecular underpinnings of priming interactions. Understanding the factors that drive these different fates could be therapeutically harnessed to reduce exhaustion phenotypes and increase effector functions and memory potential of CD8⁺ T cells in cancer.

Methods We used therapeutic interventions as a tool to alter the signals received by a T cell during priming and characterized the impact on T cell fate. Using a BRAF^V600EPTEN^-/- melanoma cell line that expresses the SIYRYYGL (SIY) model antigen, we implanted tumors and provided therapy during priming. We surveyed the effects of checkpoint blockade therapy (anti-PD1 and anti-CTLA4), costimulatory agonists (anti-CD40 and anti-41BB), and Type-I Interferon (IFNa and IFNb) on tumor-reactive CD8⁺ T cell phenotypes at early timepoints during priming via flow cytometry and single-cell RNA sequencing.

Results All treatments resulted in an increase of effector-like (TCF1^-TIM3⁺) tumor-reactive T cells. Interestingly however, each intervention induced differential expression of a specific set of transcription factors. For example, expression of Eomes is significantly increased upon IFNb treatment at both protein and RNA level, while it is decreased in T cells exposed to agonistic anti-CD40. Analysis of single-cell RNA sequencing of tumor-reactive CD8⁺ T cells revealed unsupervised clustering of cells based on the therapeutic intervention administered during priming. Tumor outgrowth and phenotypic studies at later timepoints indicated that CD8⁺ T cells primed with therapeutic intervention adopt different cell fates.

Conclusions Priming signals are crucial determinants of T cell fate. Changing the signals received by a CD8⁺ T cell during priming results in an altered phenotypic and transcriptional profile early during priming. These early transcriptional changes appear to generate different CD8⁺ T cell fates, providing an opportunity to improve anti-tumor immunity in a neoadjuvant setting.

Acknowledgements MIT Frontier Research Grant

Ethics Approval All mouse experiments in this study were approved by MIT's Committee on Animal Care (CAC) – DHHS Animal Welfare Assurance # D16-00078