ABSTRACT: Dendritic cells (DCs) are promising targets for cancer immunotherapies because of their central role in the initiation and control of immune responses. The rare cDC1 population is of particular interest because of its remarkable ability to cross-present antigens (Ag) to CD8+ T cells, to promote Th1 cell polarization and NK cell activation and recruitment. However, the spatial organization and specific functions of cDC1s in response to immunotherapy remain to be clearly characterized in human tumors. Here, we implemented a multiplexed immunofluorescence analysis pipeline coupled with computational image analysis to determine the spatial organization of the cDC1 subset in a cohort of skin lesions from advanced melanoma patients treated with immune checkpoint inhibitors (ICI). For this, we performed a whole-slide image analysis of cDC1 infiltration and distribution as well as their spatial interactions with key immune partners such as CD8+ T cells and pDC according to the response of patients to ICI. We also analyzed LAMP3+-DC, which correspond to a mature subset of tumor-infiltrating DCs. Distance and cell network analyses demonstrated that cDC1s exhibited a scattered distribution compared to tumor-infiltrating pDCs and LAMP3+-DCs, which were preferentially organized in dense areas with high homotypic connections. Interestingly, the proximity and interactions between CD8+ T cells and cDC1s were positively associated with the response to ICI. In conclusion, our study unravels the complex spatial organization of cDC1s and their interactions with CD8+ T cells in melanoma patient lesions, shedding light on their pivotal role in shaping the response to ICI.