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Background Intravesical Bacillus Calmette-Guérin (BCG) immunotherapy remains the cornerstone for managing high-risk non-muscle invasive bladder cancer (NMIBC). However, up to 40% of patients eventually fail BCG therapy due to tumor recurrence or progression. Therefore, it is critical to identify patients at risk for treatment failure before BCG initiation. These patients might be candidates for early radical cystectomy (RC), which, although negatively affecting quality of life, has excellent long-term outcomes. Additional treatment strategies are urgently needed amid an ongoing global BCG shortage. To address this need, we performed single-cell RNA sequencing (scRNA-seq) of BCG-treated NMIBC tumors to dissect the tumor microenvironment and identify cellular features associated with treatment response.Methods We performed scRNA-seq on pre-treatment tumor specimens from 10 patients with high-risk NMIBC (5 BCG-responsive and 5 BCG-unresponsive). After quality control and contamination filtering, unsupervised clustering and cell type annotation were performed. T cell subclustering was conducted to identify functional subsets, and pathway enrichment analyses were applied to determine immune and epithelial cell state differences.Results We identified 13 major cell populations in the NMIBC tumor microenvironment, including epithelial, T, B, myeloid, and plasma cells. Cell type-specific marker genes such as EPCAM, PECAM1, CD3D, CD14, COL1A1, and CD79A confirmed the annotation of each cluster. BCG-unresponsive tumors exhibited a significantly higher proportion of GZMK + CD8+ T cells compared to responders (figure 1A). Differential gene expression analysis revealed increased expression of cytotoxicity- and migration-related genes (GZMK, CXCR4) and decreased expression of exhaustion-associated markers (CXCL13, GZMB) in non-responders (figure 1B), suggesting a dysfunctional, senescence-like CD8+ T cell phenotype.Plasmacytoid dendritic cells (pDCs), a critical source of type I interferons, were reduced in the non-responder group (figure 2A). Notably, pDC abundance inversely correlated with the proportion of GZMK+ CD8+ T cells (figure 2B), and enrichment analysis indicated downregulation of antigen presentation-related pathways in non-responders (figure 2C). These findings suggest that BCG unresponsiveness may be driven by both impaired dendritic cell-mediated signaling and an aging-associated CD8+ T cell landscape.Conclusions Our single-cell transcriptomic profiling reveals that BCG-unresponsive NMIBC is characterized by dysfunctional CD8 + T cell states and impaired epithelial antigen presentation, both linked to reduced pDC presence. These alterations suggest an ‘inflammaging’ immune microenvironment that fails to effectively respond to BCG. This work highlights the importance of immune senescence and dendritic cell-mediated interferon signaling in shaping therapeutic outcomes and provides a rationale for developing combination or alternative strategies for BCG-unresponsive patients.Abstract 779 Figure 1Dysfunctional GZMK+ CD8+ T cells enriched in BCG-unresponsive tumorsAbstract 779 Figure 2Depletion of pDCs and impaired antigen presentation in non-responders