Anti-PD-1 efficacy is associated with a clonal trajectory composed of chronic effectors with lymph node homing capacity

Introduction: Immune checkpoint blockade (ICB), such as anti-CTLA-4 or anti-PD-1 (alphaPD-1), drives distinct patterns of T cell differentiation that ultimately influence treatment efficacy. However, it has been challenging to identify and define distinct T cell differentiation states in humans. We apply a novel algorithm Clonotrace (Nancy Zhang, UPenn) to define differentiation trajectories by monitoring single clones across differentiation states after alphaPD-1. We test the hypothesis that certain differentiation trajectories will have unique properties, including cell states and trafficking patterns, that will influence clinical efficacy. Methods: We performed single cell RNA and paired TCR sequencing on 132,898 CD8+ T cells from 36 stage III melanoma patients treated with neoadjuvant alphaPD-1, including 12 patients with matched tumor, uninvolved regional lymph node, and blood samples. We applied Clonotrace to define differentiation trajectories by grouping clonotypes that have a similar transcriptional footprint across CD8 cell states. We then defined these differentiation trajectories in terms of cell state composition, antigen specificity, tissue distribution, gene regulation, and association with clinical outcomes. Results: We identified 10 distinct clonal trajectories that differentiate from naïve/stem-like CD8 T cell states to exhausted and/or effector CD8 T cell states. These trajectories largely separated into two sets of clones based on antigen specificity and the expression of pathways associated with chronic stimulation. Further examination of the antigen-specific clones identified one particular trajectory (Traj 5), that was associated with clinical response. Traj 5 included clones with a unique chronic effector cell state that was able to traffic between lymph node and tumor. In contrast, Traj 1 was composed of terminally exhausted CD8 clones that were tumor-resident and associated with alphaPD-1 resistance. Conclusion: We identify a unique differentiation trajectory in patients that retains effector T cell differentiation in the setting of chronic stimulation. These chronic effectors retain the ability to traffic between lymph node and tumor and are associated with the efficacy of alphaPD-1. These findings indicate maintaining trafficking ability is essential for the efficacy of ICB. In contrast, terminal differentiation results in CD8 T cells becoming “locked” in the tissue and terminally exhausted.