Temporally resolved single-cell transcriptomics defines immune dysfunction trajectories in glioblastoma

Immune cells sense their environment and adapt their state. Deciphering cellular trajectories unfolding over time is fundamental for understanding biology. Empirical in vivo genomic technologies measuring both cell state and time are lacking. Here, we present Zman-seq, a single-cell technology tracking transcriptomic dynamics across time by introducing fluorescent time stamps into immune cells. We applied Zman-seq to resolve immune cell state-transitions of the dysfunctional tumor immune microenvironment (TME) of glioblastoma (GBM). By measuring the exposure time of infiltrating immune cells in the TME, Zman-seq defines the cellular and molecular trajectories underlying immune incompetence in GBM. Within 24 hours, cytotoxic NK cells transitioned to a dysfunctional program regulated by Tgfb1, while monocytes differentiated into immunosuppressive macrophages in 36-48 hours. Anti-TREM2 antagonistic antibody reshaped the TME by redirecting monocytes to pro-inflammatory macrophages. Zman-seq is a broadly applicable technology enabling empirical measurements of differentiation trajectories guiding the development of more efficacious immunotherapies. Overall design: Wt B6 mice were injected or not injected with GL261 cells into the right striatum. Some mice did not receive tumor. Some tumor bearing mice were treated with anti-TREM2 antibody or isotype control at days 2 and 7, some did not receive treatment. Mice were injected with fluorescent antibodies on days 11-12. On day 13 brains were extracted and single cell suspension enriched for CD45 cells were prepared. For some untreated mice blood, spleen, colon and lung tissue was also processed and CD45+ cells extracted. This was followed by single cell FACS index sorting into 384-well plates. Single cell RNAseq libraries were prepared with the MARSseq protocol and sequenced with Illumina Novaseq.