RUFY3 regulates endolysosomes perinuclear positioning, antigen presentation and migration in activated phagocytes.

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.

Peyer's patch phagocytes acquire specific transcriptional programs that influence their maturation and activation profiles.

Peyer's patches (PPs) are secondary lymphoid organs in contact with the external environment via the intestinal lumen, thus combining antigen sampling and immune response initiation sites. Therefore, they provide a unique opportunity to study the entire process of phagocyte differentiation and activation in vivo. Here, we deciphered the transcriptional and spatial landscape of PP phagocyte populations from their emergence in the tissue to their final maturation state at homeostasis and under stimulation. Activation of monocyte-derived Lysozyme-expressing dendritic cells (LysoDCs) differs from that of macrophages by their upregulation of conventional DC (cDC) signature genes such as Ccr7 and downregulation of typical monocyte-derived cell genes such as Cx3cr1. We identified gene sets that distinguish PP cDCs from the villus ones and from LysoDCs. We also identified key immature, early, intermediate, and late maturation markers of PP phagocytes. Finally, exploiting the ability of the PP interfollicular region to host both villous and subepithelial dome emigrated cDCs, we showed that the type of stimulus, the subset, but also the initial location of cDCs shape their activation profile and thus direct the immune response. Our study highlights the importance of targeting the right phagocyte subset at the right place and time to manipulate the immune response.

Viral infection engenders bona fide and bystander subsets of lung-resident memory B cells through a permissive mechanism.

Lung-resident memory B cells (MBCs) provide localized protection against reinfection in respiratory airways. Currently, the biology of these cells remains largely unexplored. Here, we combined influenza and SARS-CoV-2 infection with fluorescent-reporter mice to identify MBCs regardless of antigen specificity. We found that two main transcriptionally distinct subsets of MBCs colonized the lung peribronchial niche after infection. These subsets arose from different progenitors and were both class switched, somatically mutated, and intrinsically biased in their differentiation fate toward plasma cells. Combined analysis of antigen specificity and B cell receptor repertoire segregated these subsets into "bona fide" virus-specific MBCs and "bystander" MBCs with no apparent specificity for eliciting viruses generated through an alternative permissive process. Thus, diverse transcriptional programs in MBCs are not linked to specific effector fates but rather to divergent strategies of the immune system to simultaneously provide rapid protection from reinfection while diversifying the initial B cell repertoire.

The Salmonella effector SifA initiates a kinesin-1 and kinesin-3 recruitment process mirroring that mediated by Arl8a and Arl8b.

When intracellular, pathogenic Salmonella reside in a membrane compartment composed of interconnected vacuoles and tubules, the formation of which depends on the translocation of bacterial effectors into the host cell. Cytoskeletons and their molecular motors are prime targets for these effectors. In this study, we show that the microtubule molecular motor KIF1Bß (a splice variant of KIF1B), a member of the kinesin-3 family, is a key element for the establishment of the Salmonella replication niche as its absence is detrimental to the stability of bacterial vacuoles and the formation of associated tubules. Kinesin-3 interacts with the Salmonella effector SifA but also with SKIP (also known as PLEKHM2), a host protein complexed to SifA. The interaction with SifA is essential for the recruitment of kinesin-3 on Salmonella vacuoles whereas that with SKIP is incidental. In the non-infectious context, however, the interaction with SKIP is essential for the recruitment and activity of kinesin-3 only on a fraction of the lysosomes. Finally, our results show that, in infected cells, the presence of SifA establishes a kinesin-1 and kinesin-3 recruitment pathway that is analogous to and functions independently of that mediated by the Arl8a and Arl8b GTPases. This article has an associated First Person interview with the first author of the paper.

Author Correction: Phosphoinositides regulate the TCR/CD3 complex membrane dynamics and activation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Microtubule plus-end dynamics link wound repair to the innate immune response.

The skin protects animals from infection and physical damage. In Caenorhabditis elegans, wounding the epidermis triggers an immune reaction and a repair response, but it is not clear how these are coordinated. Previous work implicated the microtubule cytoskeleton in the maintenance of epidermal integrity (Chuang et al., 2016). Here, by establishing a simple wounding system, we show that wounding provokes a reorganisation of plasma membrane subdomains. This is followed by recruitment of the microtubule plus end-binding protein EB1/EBP-2 around the wound and actin ring formation, dependent on ARP2/3 branched actin polymerisation. We show that microtubule dynamics are required for the recruitment and closure of the actin ring, and for the trafficking of the key signalling protein SLC6/SNF-12 toward the injury site. Without SNF-12 recruitment, there is an abrogation of the immune response. Our results suggest that microtubule dynamics coordinate the cytoskeletal changes required for wound repair and the concomitant activation of innate immunity.

SAPHIR: a Shiny application to analyze tissue section images.

Study of cell populations in tissues using immunofluorescence is a powerful method for both basic and medical research. Image acquisitions performed by confocal microscopy notably allow excellent lateral resolution and more than 10 parameter measurements when using spectral or multiplex imaging. Analysis of such complex images can be very challenging and easily lead to bias and misinterpretation. Here, we have developed the Shiny Analytical Plot of Histological Image Results (SAPHIR), an R shiny application for histo-cytometry using scatterplot representation of data extracted by segmentation. It offers many features, such as filtering of spurious data points, selection of cell subsets on scatterplot, visualization of scatterplot selections back into the image, statistics of selected data and data annotation. Our application allows to characterize labeled cells, from their phenotype to their number and location in the tissue, as well as their interaction with other cells. SAPHIR is available from: https://github.com/elodiegermani/SAPHIR.

Phosphoinositides regulate the TCR/CD3 complex membrane dynamics and activation.

Phosphoinositides (PIs) play important roles in numerous membrane-based cellular activities. However, their involvement in the mechanism of T cell receptor (TCR) signal transduction across the plasma membrane (PM) is poorly defined. Here, we investigate their role, and in particular that of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] in TCR PM dynamics and activity in a mouse T-cell hybridoma upon ectopic expression of a PM-localized inositol polyphosphate-5-phosphatase (Inp54p). We observed that dephosphorylation of PI(4,5)P2 by the phosphatase increased the TCR/CD3 complex PM lateral mobility prior stimulation. The constitutive and antigen-elicited CD3 phosphorylation as well as the antigen-stimulated early signaling pathways were all found to be significantly augmented in cells expressing the phosphatase. Using state-of-the-art biophotonic approaches, we further showed that PI(4,5)P2 dephosphorylation strongly promoted the CD3ε cytoplasmic domain unbinding from the PM inner leaflet in living cells, thus resulting in an increased CD3 availability for interactions with Lck kinase. This could significantly account for the observed effects of PI(4,5)P2 dephosphorylation on the CD3 phosphorylation. Our data thus suggest that PIs play a key role in the regulation of the TCR/CD3 complex dynamics and activation at the PM.

A straightforward STED-background corrected fitting model for unbiased STED-FCS analyses.

Combining stimulated emission depletion and fluorescence correlation spectroscopy (STED-FCS) provides a powerful and sensitive tool for studying the molecular dynamics in live cells with high spatio-temporal resolution. STED-FCS gives access to molecular diffusion characteristic at the nanoscale occurring within short period of times. However due to the incomplete suppression of fluorescence in the STED process, the STED-FCS point spread function (PSF) deviates from a Gaussian shape and challenges the analysis of the auto-correlation curves obtained by FCS. Here, we model the effect of the incomplete fluorescence suppression in STED-FCS experiments and propose a new fitting model improving the accuracy of the diffusion times and average molecule numbers measurements. The implementation of a STED module with pulsed laser source on a commercial confocal/FCS microscope allowed us to apply the STED-background corrected model to fit the STED-FCS measurements. The experimental results are in good accordance with the theoretical analysis both for the number of molecules and the diffusion time which decrease accordingly with the STED power.

Protein synthesis inhibition and GADD34 control IFN-ß heterogeneous expression in response to dsRNA.

In innate immune responses, induction of type-I interferons (IFNs) prevents virus spreading while viral replication is delayed by protein synthesis inhibition. We asked how cells perform these apparently contradictory activities. Using single fibroblast monitoring by flow cytometry and mathematical modeling, we demonstrate that type-I IFN production is linked to cell's ability to enter dsRNA-activated PKR-dependent translational arrest and then overcome this inhibition by decreasing eIF2α phosphorylation through phosphatase 1c cofactor GADD34 (Ppp1r15a) expression. GADD34 expression, shown here to be dependent on the IRF3 transcription factor, is responsible for a biochemical cycle permitting pulse of IFN synthesis to occur in cells undergoing protein synthesis inhibition. Translation arrest is further demonstrated to be key for anti-viral response by acting synergistically with MAVS activation to amplify TBK1 signaling and IFN-ß mRNA transcription, while GADD34-dependent protein synthesis recovery contributes to the heterogeneous expression of IFN observed in dsRNA-activated cells.

Distinct patterns of cytolytic T-cell activation by different tumour cells revealed by Ca2+ signalling and granule mobilization.

Cancer-germline genes in both humans and mice have been shown to encode antigens susceptible to targeting by cytotoxic CD8 T effector cells (CTL). We analysed the ability of CTL to kill different tumour cell lines expressing the same cancer-germline gene P1A (Trap1a). We previously demonstrated that CTL expressing a T-cell receptor specific for the P1A35-43 peptide associated with H-2Ld , although able to induce regression of P1A-expressing P815 mastocytoma cells, were much less effective against P1A-expressing melanoma cells. Here, we analysed parameters of the in vitro interaction between P1A-specific CTL and mastocytoma or melanoma cells expressing similar levels of the P1A gene and of surface H-2Ld . The mastocytoma cells were more sensitive to cytolysis than the melanoma cells in vitro. Analysis by video-microscopy of early events required for target cell killing showed that similar patterns of increase in cytoplasmic Ca2+ concentration ([Ca2+ ]i) were induced by both types of P1A-expressing tumour cells. However, the use of CTL expressing a fluorescent granzyme B (GZMB-Tom) showed a delay in the migration of cytotoxic granules to the tumour interaction site, as well as a partially deficient GZMB-Tom exocytosis in response to the melanoma cells. Among surface molecules possibly affecting tumour-CTL interactions, the mastocytoma cells were found to express intercellular adhesion molecule-1, the ligand for LFA-1, which was not detected on the melanoma cells.

RUN and FYVE domain-containing protein 4 enhances autophagy and lysosome tethering in response to Interleukin-4.

Autophagy is a key degradative pathway coordinated by external cues, including starvation, oxidative stress, or pathogen detection. Rare are the molecules known to contribute mechanistically to the regulation of autophagy and expressed specifically in particular environmental contexts or in distinct cell types. Here, we unravel the role of RUN and FYVE domain-containing protein 4 (RUFY4) as a positive molecular regulator of macroautophagy in primary dendritic cells (DCs). We show that exposure to interleukin-4 (IL-4) during DC differentiation enhances autophagy flux through mTORC1 regulation and RUFY4 induction, which in turn actively promote LC3 degradation, Syntaxin 17-positive autophagosome formation, and lysosome tethering. Enhanced autophagy boosts endogenous antigen presentation by MHC II and allows host control of Brucella abortus replication in IL-4-treated DCs and in RUFY4-expressing cells. RUFY4 is therefore the first molecule characterized to date that promotes autophagy and influences endosome dynamics in a subset of immune cells.

Barcoding T cell calcium response diversity with methods for automated and accurate analysis of cell signals (MAAACS).

We introduce a series of experimental procedures enabling sensitive calcium monitoring in T cell populations by confocal video-microscopy. Tracking and post-acquisition analysis was performed using Methods for Automated and Accurate Analysis of Cell Signals (MAAACS), a fully customized program that associates a high throughput tracking algorithm, an intuitive reconnection routine and a statistical platform to provide, at a glance, the calcium barcode of a population of individual T-cells. Combined with a sensitive calcium probe, this method allowed us to unravel the heterogeneity in shape and intensity of the calcium response in T cell populations and especially in naive T cells, which display intracellular calcium oscillations upon stimulation by antigen presenting cells.

Peyer's patch dendritic cells sample antigens by extending dendrites through M cell-specific transcellular pores.

BACKGROUND & AIMS: Peyer's patches (PPs) of the small intestine are antigen sampling and inductive sites that help establish mucosal immunity. Luminal antigens are transported from the mucosal surface of PPs to the subepithelial dome (SED), through the specialized epithelial M cells of the follicle-associated epithelium. Among the SED resident dendritic cells (DCs), which are situated ideally for taking up these antigens, some express high levels of lysozyme (LysoDC) and have strong phagocytic activity. We investigated the mechanisms by which LysoDCs capture luminal antigens in vivo. METHODS: We performed 2-photon microscopy on explants of PPs from mice in which the enhanced green fluorescent protein gene was inserted into the lysozyme M locus (lys-EGFP mice), allowing fluorescence detection of LysoDC. RESULTS: LysoDC extended dendrites through M-cell-specific transcellular pores to the gut lumen. The M-cell adhesion molecules junctional adhesion molecule-A and epithelial cell adhesion molecule were recruited to sites of transcellular migration. Transcellular dendrites scanned the M-cell apical surface and the gut luminal content; they were able to take pathogenic bacteria and inert particles in the lumen before retracting back to the SED. CONCLUSIONS: We describe an antigen sampling mechanism that occurs in PPs and involves cooperation between M cells of the follicle-associated epithelium and DCs of the subepithelial dome. This process might be developed to target vaccines to the mucosa.

Stromal cell networks regulate thymocyte migration and dendritic cell behavior in the thymus.

After entry into thymus, T cell progenitors migrate in the cortex and the medulla while completing their education. Recent reports have documented the dynamic and tortuous behavior of thymocytes. However, other than chemokines and/or segregated thymic substrates, the factors contributing to the dynamic patterns of thymocyte movement are poorly characterized. By combining confocal and dynamic two-photon microscopy, we demonstrate that thymocytes continuously migrate on thymic stromal cell networks. In addition to constituting "roads" for thymocytes, we observed that these networks also provide a scaffold on which dendritic cells attach themselves. These results highlight the central role of stromal microanatomy in orchestrating the multiple cellular interactions necessary for T cell migration/development within the thymus.

Intracellular trafficking of LET-756, a fibroblast growth factor of C. elegans, is controlled by a balance of export and nuclear signals.

The superfamily of fibroblast growth factors (FGF), which counts 22 members in humans, exerts many functions during animal development and adult life. LET-756 is one of the two FGFs of the nematode C. elegans. Re-introduction of LET-756 in a null mutant strain restores viability, allowing the study of structural requirements for LET-756 trafficking and function. LET-756 protein has several regions and motifs, including a non-classical internal motif required for secretion. We show here that a main difference in the wild-type LET-756 molecule and a truncated molecule that mimics a partial loss-of-function mutant lies on subnuclear expression. Using Cos-1 cells and rescue activity we show that: (i) nuclear localization is due to various redundant NLS, one of them acting as a nucleolar localization signal; (ii) nuclear LET-756 is addressed to the speckles by a stretch of glutamine residues; (iii) nuclear LET-756 is trafficking between speckles and nucleoli; (iv) in the nucleolus, LET-756 is associated with proteins of the rRNA splicing compartment; (v) changing LET-756 secretion signal prevents its nuclear localization. We propose that LET-756 exerts its functions through a balance between secreted and nuclear forms due to two opposite addressing signals, (i) synergy of several NLS and (ii) attenuated secretion signal.