Spatial transcriptional dynamics of CD74⁺ B cells in tertiary lymphoid structures and effects on immune evolution in penile squamous cell carcinoma.

5038 Background: Antitumor immunity has become a critical focus in improving Penile squamous cell carcinoma (PSCC) patient outcomes. Among various immune features, tertiary lymphoid structures (TLS) have emerged as crucial players in tumor immunity. Nevertheless, the specific role of B cells within TLS and their interaction with naive T cells in PSCC remain largely unclear. Therefore, this study aims to elucidate the role of CD74⁺ B cells in TLS and their impact on immune regulation in PSCC. Methods: Spatial transcriptomics produced 519,608 cellular profiles from tumor samples of 18 patients. Additionally, scRNA-seq on tissues from 21 patients yielded 150,540 cells. Bulk RNA-seq was performed on tissues from 55 patients. TLS were identified and visualized using the DBSCAN algorithm, which clusters B cells, T cells, and DCs. Survival analyses were conducted to examine the association between TLS density and patient outcomes. Moreover, immune activity and cell infiltration were assessed via AUCell and CIBERSORT, while pseudotime analysis was used to explore dynamic gene expression changes during cell development. Additionally, pathway enrichment analysis identified key signaling pathways, and intercellular signaling was analyzed through cell communication models. Results: TLS, accurately identified using the DBSCAN algorithm, were significantly associated with improved prognosis in PSCC patients (Internal cohort, n = 152, P < 0.01; External cohort, n=63, P < 0.05). The spatial distribution, a high density of B cells was observed within the TLS regions. Notably, CD74⁺ B cells were enriched within TLS, particularly during early developmental stages. Mapped the spatially in situ developmental trajectory of CD74⁺ B cells and uncovered the dynamic changes in gene expression throughout their maturation process, we observed that CD74⁺ B cells within the TLS of PSCC patients predominantly exhibited features of early developmental stages. In line with this, scRNA-seq data further validated these observations. These cells, through their HLA molecules, interacted with CD4/CD8 ligands on naive T cells, thereby activating critical transcription factors such as NFKB1, NFKB2, NFATC1, NFATC2, FOS, and RUNX1. This interaction ultimately amplified immune responses within the tumor microenvironment. Furthermore, patients with higher CD74⁺ B cell expression exhibited better responses to immunotherapy (pCR : P < 0.01and CR: P < 0.01) and underscoring the therapeutic relevance of these cells. Conclusions: By activating naive T cells through antigen presentation, CD74⁺ B cells within TLS significantly enhance local immune responses in PSCC. Thus, CD74⁺ B cells not only serve as a promising biomarker but also represent a potential therapeutic target, providing novel insights into the immunological mechanisms underlying PSCC progression and response to immunotherapy.