Investigating the temporal dynamics of T cells following cancer immunotherapy

Abstract

The blockade of immune checkpoints PD1 and CTLA4 have become a standard treatment for the treatment of melanoma. However, mechanistic understanding of how the immunotherapy works remains to be achieved. It is largely unknown how tumour-reactive T cells regulate their checkpoint expression in vivo, and how immune checkpoint blockade regulates such activation dynamics. Capturing temporally dynamic changes that occur following T cell activation at a single cell level remains a challenge, due to limitations in methods. To address this, I used Nr4a3-Timer- of-cell-kinetics-and-activity (Tocky), which allows analysis of temporal dynamics of activated T cells following TCR signalling in vivo. Furthermore, the Tocky method is developed into a multidimensional method for analysing spectrum analyser data and single cell (sc)RNA-seq data, providing a deeper mechanistic understanding of checkpoint blockade. By analysing melanoma-bearing Nr4a3 Tocky mice, hidden dynamics of tumour-reactive T cells are elucidated in the steady-state. Checkpoint blockade depleted highly activated Treg, while promoting unique effector T cell populations, and thus differentially modulating activation of tumour-reactive T cell populations. Furthermore, multidimensional analysis and seamless analysis of Tocky and expression data revealed a full spectrum of T cell dynamics in response to tumour burden and treatment with checkpoint blockade. Lastly, we propose a rational design of combinatorial therapy to further enhance T cell activities , demonstrating a role for an OX40 agonist in combination with dual checkpoint blockade to increase effector populations and decrease regulatory T cell populations, overall increasing therapeutic benefit compared to dual blockade. Furthermore, the dynamics of Foxp3 transcription were investigated by Foxp3-Tocky, showing the dynamics of Treg in the Tumour compared to other organs. By investigating Foxp3 dynamics in Foxp3-tocky, we show that dual checkpoint blockade increases New Foxp3 transcription systemically, while being more short lived in the tumour. Lastly, the role of activation induced cell death in mediating the effects of dual checkpoint immunotherapy was investigated using BIMKO-Nr4a3-tocky mice. This showed that the immunotherapy dynamically controls the composition of antigen-reactive T cells by cell death following TCR signals.