Scalable generation of polyfunctional therapeutic virus-specific T cells with tissue-homing features and resident-memory-like plasticity across viral targets

<title>Abstract</title> <p> Adoptive transfer of virus-specific T cells (VSTs) is an effective strategy to control opportunistic viral infections, yet low precursor frequencies and manufacturing constraints limit broad implementation, particularly for viruses such as adenovirus and BK virus. Here, we established a 10-day GMP-compatible expansion strategy to generate highly specific VST products from interferon-γ-enriched cells derived from HLA-matched donors, suitable for cryobanking and multi-dose administration. Compared with expansion from whole leukapheresis, initial enrichment of interferon-γ secreting cells limited NK cell and non-specific T cell outgrowth, and restricted the expansion of bystander activated T cells. Moreover, omission of additional peptide restimulation during production better maintained CD4/CD8 composition and chemokine receptor profiles compatible with tissue-homing potential, while preserving activation- and cytokine-related transcriptional programs. Expanded VSTs across viral targets retained a predominantly effector-memory phenotype, displayed virus-directed polyfunctional cytokine production, and mediated antigen-specific cytotoxicity without detectable reactivity against allogeneic cells. TCRβ repertoire analyses revealed clonal focusing in expanded VSTs while maintaining multi-epitope specificity. Scaled-up manufacturing yielded more than 10⁸ BK virus-specific T cells with high antigen-specific functionality despite low initial precursor frequencies, with specificity preserved after cryopreservation. Notably, after 3 days of exposure to cognate antigen and TGF-β1, VSTs upregulated tissue-resident memory T cell (T _RM ) markers CD69 and CD103, while retaining antiviral cytokine production above unstimulated levels, indicating resident-memory-like phenotypic plasticity. These data support a rapid, scalable platform to generate allogeneic VST products with high specificity, tissue-homing features, and T _RM -like plasticity for the treatment of refractory viral complications and future engineered-cell applications. </p>