AbstractCD8⁺ T cell reactivity towards tumor mutation-derived neoantigens is widely believed to facilitate the antitumor immunity induced by immune checkpoint blockade (ICB). Here we show that broadening in the number of neoantigen-reactive CD8⁺ T cell (NART) populations between pre-treatment to 3-weeks post-treatment distinguishes patients with controlled disease compared to patients with progressive disease in metastatic urothelial carcinoma (mUC) treated with PD-L1-blockade. The longitudinal analysis of peripheral CD8⁺ T cell recognition of patient-specific neopeptide libraries consisting of DNA barcode-labelled pMHC multimers in a cohort of 24 patients from the clinical trial NCT02108652 also shows that peripheral NARTs derived from patients with disease control are characterised by a PD1⁺ Ki67⁺ effector phenotype and increased CD39 levels compared to bystander bulk- and virus-antigen reactive CD8⁺ T cells. The study provides insights into NART characteristics following ICB and suggests that early-stage NART expansion and activation are associated with response to ICB in patients with mUC.