Towards a molecular understanding of centrosome integrity, function and organization in dendritic cells during T cell priming

The centrosome, nucleating microtubules (MTs) and serving as primary microtubule-organizing center (MTOC), is essential for eukaryotic cell architecture. It is composed of two centrioles surrounded by pericentriolar material (PCM). The centrosome's major functions include enabling cell division, regulating intracellular organization, and facilitating cell polarity and signaling – several functions being important, for instance, in immune cells. For the initiation of adaptive immune responses, dendritic cells (DCs), which are professional antigen-presenting cells, form specialized cell-cell contacts with T cells. During immune synapse (IS) formation, interaction of surface molecules and the supply of soluble mediators instruct T cell activation and, importantly, centrosome polarization in T cells is critical for efficient T cell priming. However, surprisingly little is known about the MT cytoskeleton within DCs during IS formation. This study investigates the role of centrioles and MTs within DCs and their impact on cellular function, particularly in the context of the induction of CD4+ T cell activation and proliferation. Through various drug treatments, we have uncovered that both components of the cytoskeleton – centrioles and MTs – are important for T cell priming. While most non-proliferating cells contain two centrioles, previous research from our group has revealed that DCs acquire elevated numbers of centrioles (>2) upon immune activation. This phenomenon, although commonly observed in cancer cells, is rare in non-malignant cells. Functionally, extra centrioles form a single over-active MTOC, characterized by increased PCM and MT nucleation. Here, we showed that cells with multiple centrioles exhibit a higher capacity to activate T cells. To understand the underlying processes, we visualized spatio-temporal dynamics of centrioles in DCs upon antigen-specific T cell contacts. We observed that multiple centrioles tightly cluster during DC-T cell interactions. Additionally, unlike in T cells, MTOC polarization was absent in DCs. Instead, DC centrioles remained centrally located in the cell, in close proximity to the nucleus. Three-dimensional visualization of DC-T cell conjugates in peripheral lymph nodes confirmed these findings in vivo. In further experiments, pharmacological perturbation of centriole clusters in DCs resulted in diminished T cell activation. The secretion of soluble mediators has been described as intracellular process linked to the MT cytoskeleton. Thus, a comprehensive secretome analysis of DCs with distinct centriole numbers was performed. The results indicated that the secretory profile of DCs differs between cells with two and multiple centrioles. Given that DC secretion largely impacts the microenvironment of other immune and non-immune cells, these findings provoke further questions regarding the response of surrounding cells. Altogether, our results highlight the crucial role of centrosome integrity and proper MT array organization in DCs for T cell activation. The acquisition of multiple centrioles and their optimal spatial configuration and positioning within DCs facilitate the efficient initiation of adaptive immune responses. Future studies elucidating the impact of centrosome or MT array alterations will substantially advance our understanding of immune cell functionality and may facilitate the development of novel therapeutic strategies for example in the context of cancer treatment.