Energetic metabolism reprogramming is critical for cancer and immune responses. Current methods to functionally profile the global metabolic capacities and dependencies of cells are performed in bulk. We designed a simple method for complex metabolic profiling called SCENITH, for Single Cell ENergetIc metabolism by profilIng Translation inHibition. SCENITH allows for the study of metabolic responses in multiple cell types in parallel by flow cytometry. SCENITH is designed to perform metabolic studies ex vivo, particularly for rare cells in whole blood samples, avoiding metabolic biases introduced by culture media. We analyzed myeloid cells in solid tumors from patients and identified variable metabolic profiles, in ways that are not linked to their lineage nor their activation phenotype. SCENITH ability to reveal global metabolic functions and determine complex and linked immune-phenotypes in rare cell subpopulations will contribute to the information needed for evaluating therapeutic responses or patient stratification.

Intracellular trafficking is essential for cell structure and function. In order to perform key tasks such as phagocytosis, secretion or migration, cells must coordinate their intracellular trafficking, and cytoskeleton dynamics. This relies on certain classes of proteins endowed with specialized and conserved domains that bridge membranes with effector proteins. Of particular interest are proteins capable of interacting with membrane subdomains enriched in specific phosphatidylinositol lipids, tightly regulated by various kinases and phosphatases. Here, we focus on the poorly studied RUFY family of adaptor proteins, characterized by a RUN domain, which interacts with small GTP-binding proteins, and a FYVE domain, involved in the recognition of phosphatidylinositol 3-phosphate. We report recent findings on this protein family that regulates endosomal trafficking, cell migration and upon dysfunction, can lead to severe pathology at the organismal level.

Energetic metabolism (EM) reprogramming is critical for both cancer and immune response initiation. Current methods for EM monitoring are performed in bulk and do not have the sensitivity nor the resolution to dissect EM at single cell scale. We designed a novel EM profiling method called ZENITH, that provides single-cell resolution using multiparametric flow cytometry. ZENITH highlights the dichotomy among EM displayed respectively by naïve, effector and memory T cells, demonstrating its ability to discriminate individual cell subsets on the basis on their specific metabolic status. Tumor-associated myeloid cells isolated from biopsies are also shown to display highly variable EM profiles, in ways that are not linked to their lineage nor their activation phenotype. ZENITH ability to determine complex and linked metabolic activities in discrete cell subpopulations within tissue or blood draws will contribute to the functional information needed for evaluating therapeutic responses or patient stratification.

Cells perceive overtime complex sequences of receptor stimulation that they integrate to mount an appropriate response. Yet, the influence of signal dynamics on cell responses has been poorly characterized due to technical limitations. Here, we present a generalizable approach to control receptor stimulation on unmodified primary cells. Indeed, for applications on primary murine T cells, we have engineered the LiTe system, a new recombinant optogenetics-based Light-inducible T cell engager which allows tunable and reversible spatiotemporal control of the T Cell Receptor (TCR) stimulation. We also provided in vitro evidence that this system enables efficient T cell activation with light, leading to cytokine secretion or tumor cell killing. Using specific time-gated stimulations, we have been able to orient the outcome of the activation of T cells. Overall, the LiTe system constitutes a versatile ON/OFF molecular switch allowing to decipher the cellular response to stimulation dynamics. Its original control over T cell activation opens new avenues for future precision cancer immunotherapy.

Endo-lysosomes transport along microtubules and clustering in the perinuclear area are two necessary steps for microbes to activate specialized phagocyte functions. We report that RUN and FYVE domain-containing protein 3 (RUFY3) exists as two alternative isoforms distinguishable by the presence of a C-terminal FYVE domain and by their affinity for phosphatidylinositol 3-phosphate on endosomal membranes. The FYVE domain-bearing isoform (iRUFY3) is preferentially expressed in primary immune cells and up-regulated upon activation by microbes and Interferons. iRUFY3 is necessary for ARL8b + /LAMP1+ endo-lysosomes positioning in the pericentriolar organelles cloud of LPS-activated macrophages. We show that iRUFY3 controls macrophages migration, MHC II presentation and responses to Interferon-γ, while being important for intracellular Salmonella replication. Specific inactivation of rufy3 in phagocytes leads to aggravated pathologies in mouse upon LPS injection or bacterial pneumonia. This study highlights the role of iRUFY3 in controlling endo-lysosomal dynamics, which contributes to phagocyte activation and immune response regulation.

Endo-lysosomes transport along microtubules and clustering in the perinuclear area are two necessary steps for microbes to activate specialized phagocyte functions. We report that RUN and FYVE domain-containing protein 3 exists as two alternative isoforms distinguishable by the presence of a C-terminal FYVE domain and by their affinity for PtdIns(3)P on endosomal membranes. The FYVE domain-bearing isoform (iRUFY3) is preferentially expressed in immune cells and up-regulated upon activation by microbes and cytokines. iRUFY3 is necessary for ARL8b+/LAMP1+ endo-lysosomes positioning in the pericentriolar organelles cloud of LPS-activated macrophages. We show that iRUFY3 controls macrophages migration, MHC II presentation and responses to Interferon-γ while being required for Intracellular Salmonella replication. Specific inactivation of Rufy3 in phagocytes leads to aggravated pathologies in mouse upon LPS injection or bacterial pneumonia. This study highlights iRUFY3 function, as a novel modulator of immunity, controlling endo-lysosomes dynamics, and ultimately regulating the function of activated phagocytes.

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