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Background Dendritic cells (DCs), including both monocyte-derived DCs (moDCs) and naturally circulating DCs, have been investigated as immunotherapeutic agents in both metastatic and adjuvant cancer settings. DC-based vaccines (DC Vax) are capable of eliciting tumor-specific immune responses; however, their clinical efficacy remains limited and inconsistent. To uncover immune correlates of response to moDC vaccination in metastatic melanoma, we conducted a comprehensive multi-omics analysis of the initial apheresis product and the final DC Vax formulation.Methods We employed a multi-layered analytical approach integrating multiparametric flow cytometry, single-cell RNA sequencing, and bulk transcriptomic profiling. Specifically, we analyzed:the peripheral immune profile of 21 patients with advanced melanoma undergoing moDC-based vaccination;the final DC Vax product (scRNAseq n=7; Flow Cytometry n=16);gene expression profiles of CD14+ monocytes isolated from the apheresis product (n=21).This strategy aimed to characterize the immune cell subsets and transcriptional features associated with clinical response.Results Among all cellular components analyzed, the most striking and unexpected finding was the presence of CD19 + B cells in the final DC Vax formulation. This feature significantly distinguished Responders from Non-Responders. The abundance of B cells in the vaccine positively correlated with their baseline frequency in peripheral blood. Notably, within the DC Vax, B cells displayed a CD20^lowCD27^hiCD38^hi plasmablast-like phenotype, distinct from their autologous circulating counterparts. In parallel, tumor biopsies collected prior to treatment from Responders showed marked B cell enrichment, often organized as Tertiary Lymphoid Structures (TLS), suggesting a coordinated systemic and local B cell response associated with clinical benefit.Conclusions Our findings highlight a previously underappreciated role for B cells in both the baseline immune context and the final composition of DC-based vaccines. The presence and functional phenotype of B cells may critically influence vaccine efficacy, potentially mirroring the collaborative interactions between DC and B cell within TLS. These insights offer a novel perspective on the mechanisms underlying DC-based immunotherapy and may guide the development of more effective vaccine strategies.Acknowledgements M.T. M.B. and M.M.T. acknowledges support from the ‘Associazione Italiana per la Ricerca sul Cancro’ (grant no. MFAG 2021—ID. 26339 to M.T.). All of the other authors with IRCCS IRST affiliation acknowledge support from the Italian Ministry of Health and the contribution of ‘Ricerca Corrente’ within the research line ‘Precision Medicine, Gender, Ethnicity, and Geroscience: Genetic-Molecular Mechanisms in the Development, Characterization, and Treatment of Tumors.’Ethics Approval The protocol was approved by the institutional Medical Ethical Review Board (Protocol IRSTB134, cod. L3P2729, Approval date from the Romagna Ethics Committee (CEROM) 15/04/2022 and Protocol IRSTB137, cod. L3P2767, Approval date from the Romagna Ethics Committee (CEROM) 14/03/2024) and the study was conducted in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and later versions.