Background: We characterised the immune response in human prostate cancer (PC) to high dose rate brachytherapy (HDRBT) using serial pre- and post-HDRBT biopsies. Methods: Prostate biopsies and peripheral blood samples were obtained from 9 men with clinically localised PC at baseline and again 14 days following a 10 Gy fraction of HDRBT. Immune infiltrates were characterised using multiplex immunohistology (T cell and pan-immune panels) and targeted gene expression profiling (Nanostring pan-cancer immune panel). T cell repertoire was assessed using T cell receptor (TCR) Vbeta CDR3 deep sequencing. Results: HDRBT induced profound but heterogenous immune responses. At the extremes, one patient had an extensive tumor-infiltrating lymphocyte (TIL) response post-HDRBT, comprising T cells (CD4⁺> CD8⁺> Treg), B cells, and PDL1⁺ antigen presenting cells (macrophages and CD11c+ dendritic cells); another had a substantial baseline immune infiltrate eliminated by HDRBT. TIL-hi cases (either before or after HDRBT) had a conserved signature of T cell activation, differentiation, and trafficking, with upregulation of checkpoint molecules PD1, IDO1, BTLA, CTLA4, ICOS, B7-H3 and FoxP3. Those TIL-hi specifically in response to HDRBT also had down-regulation of genes regulating myeloid differentiation and granulocyte chemotaxis pre-HDBRT. Conversely, a monocyte-like myeloid-derived suppressor cell signature was seen in the TIL-lo responders. The median number of dominant clones ( > 1% prevalence) determined by TCR sequencing was 24.5 and 26.5 pre- and post-HDRBT respectively. Only 2 clones (in 1 patient) were present at both time points with the T cell repertoire otherwise being completely new following HDRBT. A subset of PC-associated T cell dominant clones was also present in the PB in all patients, suggesting HDRBT can induce systemic immunity. Conclusions: We have shown that treatment of localized prostate cancer with HDRBT induces a marked clonal TIL infiltrate dominated by T cells likely to be operating under the control of multiple checkpoints. These first-in-human data may be able to inform the rational selection of immunotherapy to combine with HDRBT.