Abstract BACKGROUND A human leukocyte antigen (HLA)-A*02-restricted tumor associated antigen in the neuroligin 4 X-linked (NLGN4X) protein was found to be specifically overexpressed in human gliomas. Individualized multipeptide vaccination targeted NLGN4X and induced specific cytotoxic T cells responses in patients with newly diagnosed glioblastoma. MATERIAL AND METHODS Post vaccination NLGN4X-tetramer-sorted T cells were subjected to single cell T cell receptor (TCR) sequencing for TCR discovery. The identified TCR was delivered to human T cells (NLGN4X-TCR-T) and functional profiling was performed by flow cytometry and in vitro cytotoxicity assays. NOD scid gamma (NSG) major histocompatibility complex (MHC) I/II knockout (KO) (NSG MHC I/II KO) mice were challenged with NLGN4X-expressing experimental gliomas and treated with intracerebroventricular injection of NLGN4X-TCR-T to assess its therapeutic potential. RESULTS We apply for the first time for therapeutic use of an HLA-A*02 restricted vaccine-induced TCR that binds to the NLGN4X antigen. We show the cytotoxic and polyfunctional phenotype of NLGN4X-TCR-T in various cellular models. Intracerebroventricular delivery of NLGN4X-TCR-T prolongs survival and leads to objective radiographic responses in experimental gliomas-bearing NSG MHC I/II KO mice. CONCLUSION NLGN4X-TCR-T demonstrates efficacy in a preclinical experimental glioblastoma model. On a global scale, we provide first evidence for the therapeutic retrieval of vaccine-induced human TCRs for the off-the-shelf treatment of glioblastoma patients.