Biology subjects

Raimondi, A.

Publications and source records attributed to Raimondi, A..

At least 19 recordsLinked to original sources

bioRxiv2026

A reversible temperature block captures pre-lysosomal intermediates of starvation-induced ERphagy

The selective lysosomal turnover of endoplasmic reticulum (ER) portions, termed ERphagy, removes aged, excess or damaged ER domains. The intermediates forming between receptor activation and lysosomal delivery are short-lived and remain poorly resolved. Here we identify a reversible temperature block at 25{degree sign}C that arrests ERphagy before lysosomal delivery. At this temperature, nutrient restriction still drives focal accumulation of the endogenously Halo-tagged ERphagy receptor TEX264 (puncta formation), segregation of ER cargo, remodelling of the ER membrane, whereas delivery of this material to degradative endolysosomes is strongly reduced. Rewarming above 27{degrees} restores lysosomal delivery, showing that the accumulated eTEX264-Halo puncta correspond to pre-lysosomal intermediates of the ERphagy program. Their formation requires ULK1/2 activity, the ATG7-dependent conjugation machinery, ATG9A, ATG13 and FIP200, coupling receptor clustering to autophagosome biogenesis. Consistently, expansion microscopy and electron tomography reveal curved, cradle-like TEX264-positive ER domains containing ER cargo, co-localizing with autophagy markers and lying adjacent to p62-cargo condensates.

cell biology↗

bioRxiv2026

Epigenetic evolution of colorectal cancer and its microenvironment reveals new vulnerabilities

Epigenetics is central to tumorigenesis, but the co-evolution of the cancer epigenome and its microenvironment is severely understudied. Here, we measure chromatin accessibility and transcriptome, at single cell resolution, of a set of normal colon, primary colorectal cancers and metastases, and identify recurrent epigenetic alterations in tumour cells. We also found that the normal epithelium adjacent to the cancer had recurrent epigenetic alterations associated with inflammatory programs that were partially shared with tumour cells. Distinct tumour-intrinsic transcription factor binding programs were associated with differential abundance of malignant stroma cell identities. We then leveraged matched patient-derived organoids, to assess the functional impact of the most recurrent epigenetic alterations on cancer cell viability, using CRISPR interference. We found a set of epigenetic-driven cancer dependencies, related to developmental reprogramming and cellular stress resilience, representing new potential therapeutic targets.

cancer biology↗

bioRxiv2026

Decoding EGFR ligand bias through an endocytic organelle platform

How growth factor receptors decode ligand identity into distinct cellular responses remains a fundamental question in cell signaling. Here, we identify a receptor-proximal mechanism that links ligand-specific EGFR activation to distinct endocytic and biological outputs. We show that EGF, but not TGF, selectively engages a RAC1-PLC{gamma}2-IP3R signaling axis that supports EGFR non-clathrin endocytosis (NCE). PLC{gamma}2, but not PLC{gamma}1, localizes to RTN3-dependent PM-ER contact sites, where it generates localized Ca{superscript 2} signals required for completion of NCE, mitochondrial activation and cell motility. This specificity requires the RAC-binding interface of PLC{gamma}2 and is associated with RAC1-dependent formation of CTxB-positive PM regions, indicating that spatial organization contributes to signaling specificity. TGF fails to efficiently assemble the EGFR-associated organelle platform and instead favors clathrin-dependent EGFR uptake, prolonged proliferative signaling, greater organoid yield, and reduced migration compared with EGF. Together, our findings identify the RAC1-PLC{gamma}2 axis as the key determinant that decodes EGFR ligand bias by coupling receptor trafficking to the metabolic program that supports cell migration.

cell biology↗

bioRxiv2026

Epstein Barr virus exploits ER stress and TMX4 dependent nuclear envelope remodeling to enable capsid egress

Herpesvirus capsids assemble within the nucleoplasm of infected cells in highly ordered icosahedral structures with a diameter of about 100nm. Despite the small distance between outer and inner nuclear membrane, which is fixed between the 25 and the 50nm by disulfide bonded LINC complexes, the capsid particles cross the barrier and are delivered into the cytoplasm, where viral particle assembly continues. How Epstein-Barr virus (EBV) overcomes the spatial constraints imposed by the narrow perinuclear space for nuclear egress of the viral capsids remains unclear. Here, we show that EBV exploits an ER-stress-responsive nuclear envelope (NE) remodeling pathway to promote capsid egress. Induction of EBV lytic replication activates the IRE1 branch of the unfolded protein response and triggers TMX4-dependent remodeling of the NE. Inhibition of IRE1 signaling or depletion of TMX4 prevents efficient redistribution of viral capsid proteins from the nucleus to the cytoplasm, causes accumulation of unused viral glycoprotein GP350 in Golgi-derived membranes, and markedly reduces production of infectious viral particles. Thus, EBV hijacks a host NE adaptation pathway to overcome a fundamental physical barrier during viral maturation.

cell biology↗

bioRxiv2026

Circulating extracellular vesicles drive microglial senescence and neurodegeneration in Parkinson`s disease

BackgroundExtracellular vesicles (EV), secreted membrane particles involved in cell-to-cell communication, carry important information on immunity and its dysregulation. Recent studies have demonstrated the crucial role of peripheral and central inflammation in causing Parkinsons disease (PD), as well as the involvement of EV in mediating neuron-glial interactions during neurodegeneration. However, the underlying mechanism of plasmatic EV in PD pathogenesis remains unknown. MethodsEV were isolated from pool of plasma of PD patients and age- and sex-matched healthy controls (HC) using size-exclusion chromatography and characterized by nanoparticle tracking analysis, western blot, and transmission electron microscopy. SH-SY5Y neurons and HMC3 microglia cells were treated with EV, and their impact was evaluated using flow cytometry and immunofluorescence. Conditioned medium (CM) from EV-treated HMC3 cells was applied to SH-SY5Y neurons to determine indirect neurotoxic effects. Cytokine profiling and senescence-like features of EV-treated HMC3 cells were assessed. Unbiased proteomic analysis of PD-EV and HC-EV were further performed. ResultsPD-EV induced axonal degeneration and cell death in SH-SY5Y neurons and increased levels of TNF-, IL-1{beta}, IFN-{gamma}, IL-8, and CCL11, accompanied by the expression of p16INK4a in HMC3 cells, suggesting a proinflammatory, senescence-associated secretory phenotype (SASP). Enrichment pathway analysis revealed that these changes were mainly related to inflammatory and immune responses. Moreover, CM from PD-EV-HMC3 cells increased apoptotic cell death in SH-SY5Y neurons more than direct PD-EV. Notably, proteomic analysis of PD-EV showed higher expression of proteins involved in complement cascades, immune response, phagocytosis, and post translational protein translation, further supporting the potential of EV to induce inflammatory changes in PD. ConclusionsThis study demonstrates that plasmatic PD-EV contributes to neuronal degeneration by reducing neuronal integrity and indirectly by activating microglia through the secretion of pro-inflammatory, senescence-associated mediators. Circulating EV exerts a role in bridging peripheral inflammation with microglia, modulating neuroinflammatory events.

neuroscience↗

bioRxiv2026

Targeting GL-Lect driven endocytosis to suppress cell plasticity in breast cancer

Aberrant endocytosis has long been associated with epithelial plasticity and tumorigenesis, but direct in vivo evidence of its causal role in tumor progression and metastasis has been lacking. Here, we identify and molecularly characterize a previously unrecognized form of E-cadherin (ECAD) internalization in mammary epithelial cells. This process is mediated by the endocytic adaptor Epsin 3 (EPN3) through glycolipid-lectin (GL-Lect) driven endocytosis requiring galectin-3 and Eps15-family adaptors. Leveraging an EPN3 knock-in mouse model, we show that dysregulation of GL-Lect driven endocytosis disrupts mammary gland morphogenesis and activates epithelial-to-mesenchymal plasticity (EMP), synergizing with the ERBB2/Neu breast oncogene to drive metastasis. Pharmacologic inhibition of the GL-Lect mechanism suppresses morphogenetic and invasive phenotypes ex vivo, providing proof-of-concept for therapeutic targeting. These findings establish the GL-Lect mechanism as a driver of metastatic plasticity and uncover a tractable vulnerability in BC.

cancer biology↗

bioRxiv2025

ER-phagy and mitophagy programs in human CD4+ memory T cells before and after activation

T lymphocytes can remain in a resting state for very long times. Encountering their cognate antigen triggers their activation, leading to an increase in cell size, proliferation, enhanced protein production, and secretion of signaling molecules. This activation and clonal expansion phase is followed by a contraction phase, with a reduction in cell size, eventually leading to cell death or progression to resting (memory) cells. Here, we show that human resting primary CD4+ memory T cells degrade endoplasmic reticulum (ER) and mitochondrial portions via ER-phagy and mitophagy, respectively. The temporary interruption of ER and mitochondria turnover coupled with a transient induction of the unfolded protein response results in the enlargement of the ER and cellular volume. Cell growth is stabilized and then reversed during the clonal expansion and contraction phases by the resumption of a catabolic phase characterized by the reactivation of ER-phagy and outer mitochondrial membrane (OMM)-phagy. Collectively, our data show that the maintenance and the activity of human CD4+ memory T cells rely on finely tuned execution of anabolic and catabolic programs that regulate mass and activity of the ER and mitochondria.

cell biology↗

bioRxiv2025

IL-16 production is a mechanism of resistance to BTK inhibitors and R-CHOP in lymphomas

Introducing Brutons tyrosine kinase (BTK) inhibitors has significantly improved outcomes for patients with B-cell malignancies and autoimmune disorders. However, resistance, either primary or acquired, remains a major clinical challenge. To better understand the underlying resistance mechanisms to BTK inhibitors, we established an ibrutinib-resistant model from a patient-derived splenic marginal zone lymphoma (MZL) cell line (VL51) through prolonged drug exposure. Resistant cells exhibited a 15-fold increase in ibrutinibs IC50, along with distinct morphological changes, mitochondrial activation, and cross-resistance to covalent, non-covalent BTK inhibitors and BTK degraders. Integrated transcriptomic, epigenomic, and proteomic analyses identified overexpression and secretion of IL-16 as a key feature of resistance, driven by chromatin remodeling and activation of the FLI1 transcription factor. IL-16 conferred ibrutinib resistance via CD9-mediated activation of the NF-{kappa}B and AKT signaling pathways and was found to be elevated in the serum of ibrutinib-refractory CLL patients. Functional studies showed that targeting the IL-16/CD9 axis using neutralizing antibodies or CD9-binding peptides restored sensitivity to BTK inhibitors and R-CHOP chemotherapy in MZL, mantle cell lymphoma, and diffuse large B-cell lymphoma models. These findings reveal a novel, targetable resistance mechanism with potential therapeutic implications for overcoming BTK inhibitor resistance in B-cell lymphomas.

cancer biology↗

bioRxiv2025

Tattoo ink induces inflammation in the draining lymph node and impairs the immune response against a COVID-19 vaccine

Despite safety concerns regarding the toxicity of tattoo ink, no studies have reported the consequences of tattooing on the immune response. In this work, we have characterized the transport and accumulation of different tattoo inks in the lymphatic system using a murine model. Upon quick lymphatic drainage, we observed that macrophages mainly capture the ink in the lymph node (LN). An initial inflammatory reaction at local and systemic levels follows ink capture. Notably, the inflammatory process is maintained over time as we observed clear signs of inflammation in the draining LN two months following tattooing. In addition, the capture of ink by macrophages was associated with the induction of apoptosis in both human and murine models. Furthermore, the ink accumulated in the LN altered the immune response against a COVID-19 vaccine. We observed a reduced antibody response following vaccination with a mRNA-based SARS-CoV-2 vaccine, which was associated with a decreased expression of the Spike protein in macrophages in the draining LN. Considering the unstoppable trend of tattooing in the population, our results are crucial in informing the toxicology programs, policymakers, and the general public regarding the potential risk of the tattooing practice associated with an altered immune response.

immunology↗

bioRxiv2024

The perineurium integrates leptin with its sympathetic outflow to protect against obesity

The regulatory mechanism of leptins afferent action in the brain, constituting a negative feedback loop, is contingent upon the efferent sympathetic innervation of white and brown adipose tissues. Nonetheless, the peripheral regulation governing the relative strengths of the afferent and efferent arms remains ambiguous. Using single-cell RNA sequencing on murine sympathetic ganglia, we identified the unique expression of both the leptin receptor (LepR) and the beta 2 adrenergic receptor (Adrb2) in perineurial cells that form a barrier around sympathetic ganglia and nerve bundles in adipose tissues. We show that LepR+ Sympathetic Perineurial Cells (SPCs) are molecularly similar to endothelial cells and that conditional knockout of Adrb2 in LepR+ SPCs predisposes mice to obesity without affecting food intake. Notably, we found that hyperleptinemia associated with obesity causes apoptosis in SPCs, leading to a significant erosion of the perineurial barrier and concomitant adipose sympathetic neuropathy. We further show that this deleterious effect can be reversed by sympathomimetic beta 2 adrenergic receptor agonism. These results have relevance to human obesity, as we observed a synergistic effect of highly common polymorphisms of LEPR and ADRB2 on the risk of increased BMI in a large European population. We propose that SPCs are the nexus of leptin action by integrating the afferent and efferent arms of the neuroendocrine loop to influence its setpoint.

neuroscience↗

bioRxiv2024

ER-phagy Receptors Intrinsically Disordered Modules Drive ER Fragmentation and ER-phagy

Membrane remodeling leading to fragmentation is crucial for autophagy programs that control capture by phagophores or endolysosomes of portions of organelles to be removed from cells. It is driven by membrane-bound autophagy receptors that display cytoplasmic intrinsically disordered modules (IDRs) engaging Atg8/LC3/GABARAP (LC3). Studies on endoplasmic reticulum (ER)-phagy receptors of the FAM134 family revealed the importance of sequential FAM134 proteins phosphorylation, ubiquitylation and clustering for execution of the ER-phagy programs. In this model, ER fragmentation is promoted/facilitated by the membrane-remodeling function of FAM134 reticulon homology domains (RHDs). However, RHDs are not conserved in ER-phagy receptors. The question that we tackle in this work is if activation of ER-phagy receptors anchored at the ER membrane with conventional membrane spanning domains, i.e., most of the ER-phagy receptors known to date, eventually trigger ER remodeling and fragmentation, and how. Here, we show that the membrane-tethering modules of ER-phagy receptors (RHDs for FAM134B, single/multi spanning transmembrane domains for TEX264 and SEC62) determine the sub-compartmental distribution of the receptors but are dispensable for ER fragmentation, regardless of their propensity to remodel the ER membrane. Rather, ER fragmentation is promoted by the ER-phagy receptors intrinsically disordered region (IDR) modules that are a conserved feature of all ER-phagy receptors exposed at the cytoplasmic face of the ER membrane. Since cytoplasmic IDRs with net negative charge are conserved in autophagy receptors at the limiting membrane of other organelles, we anticipate that conserved mechanisms of organelle fragmentaVon driven by cytoplasmic exposed IDRs could operate in eukaryoVc cells.

cell biology↗

bioRxiv2024

A tripartite organelle platform links growth factor receptor signaling to mitochondrial metabolism

One open question in the biology of growth factor receptors is how a quantitative input (i.e., ligand concentration) is decoded by the cell to produce specific response(s). Here, we show that an EGFR endocytic mechanism, non-clathrin endocytosis (NCE), which is activated only at high ligand concentrations and targets receptor to degradation, requires a tripartite organelle platform involving the plasma membrane (PM), endoplasmic reticulum (ER) and mitochondria. At these contact sites, EGFR-dependent, ER-generated Ca2+ oscillations are sensed by mitochondria, leading to increased metabolism and ATP production. Locally released ATP is required for cortical actin remodeling and EGFR-NCE vesicle fission. The same biochemical circuitry is also needed for an effector function of EGFR, i.e., collective motility. The multiorganelle signaling platform herein described mediates direct communication between EGFR signaling and mitochondrial metabolism, and is predicted to have a broad impact on cell physiology as it is activated by another growth factor receptor, HGFR/MET.

cell biology↗

bioRxiv2023

Simultaneous detection of membrane contact dynamics and associated Ca2+ signals by reversible chemogenetic reporters

Membrane contact sites (MCSs) are hubs allowing various cell organelles to coordinate their activities. The dynamic nature of these sites and their small size hinder analysis by current imaging techniques. To overcome these limitations, we here designed a series of reversible chemogenetic reporters incorporating improved, low-affinity variants of splitFAST, and studied the dynamics of different MCSs at high spatiotemporal resolution, both in vitro and in vivo. We demonstrated that these versatile reporters suit different experimental setups well, allowing one to address challenging biological questions. Using these novel probes, we identified a hitherto unknown pathway, in which calcium (Ca2+) signalling dynamically regulates endoplasmic reticulum-mitochondria juxtaposition, characterizing the underlying mechanism. Finally, by integrating Ca2+-sensing capabilities into the splitFAST technology, we introduced PRINCESS (PRobe for INterorganelle Ca2+-Exchange Sites based on SplitFAST), an unprecedented class of reporters to simultaneously detect MCSs and measure the associated Ca2+ dynamics using a single biosensor.

cell biology↗

bioRxiv2023

Addressing heterogeneity in direct analysis of Extracellular Vesicles and analogues using Membrane-Sensing Peptides as Pan-Affinity Probes

Extracellular vesicles (EVs), crucial mediators of cell-to-cell communication, hold immense potential for diagnostic applications due to their ability to enrich protein biomarkers in body fluids. However, challenges in isolating EVs from complex biological specimens hinder their widespread use. In this frame, integrated isolation-and-analysis workflows are the go-to strategy, most of which see the prevalence of immunoaffinity methods. Yet, the high heterogeneity of EVs poses challenges, as proposed ubiquitous markers are less homogenously prevalent than believed, raising concerns about the reliability of downstream biomarker discovery programs. This issue extends to the burgeoning field of engineered EV-mimetics and bio-nanoparticles, where conventional immune-affinity methods may lack applicability. Addressing these challenges, we introduce the use Membrane Sensing Peptides (MSP) as "universal" affinity ligands for both EVs and EV-analogues. Employing a streamlined process integrating on-bead capture and vesicle phenotyping through Single Molecule Array (SiMoA) technology, we showcase the application of MSP ligands in the integrated analysis of circulating EVs in blood derivatives, eliminating the need for prior EV isolation. Demonstrating the possible clinical translation of MSP technology, we directly detect an EV-associated epitope signature in serum and plasma samples, demonstrating its potential for distinguishing patients with myocardial infarction versus stable angina. At last, notably, MSP exhibits a unique capability to enable the analysis of tetraspanin-lacking Red Blood Cell derived EVs (RBC-EVs). Overall, unlike traditional antibody-based methods, MSP probes work agnostically, overcoming limitations associated with surface protein abundance or scarcity. This highlights the potential of MSP in advancing EV analysis for clinical diagnostics and beyond. Of note, this represents also the first-ever peptide-based application in SiMoA technology.

biochemistry↗

bioRxiv2023

EZH2 synergizes with BRD4-NUT to drive NUT carcinoma growth through silencing of key tumor suppressor genes

NUT carcinoma (NC) is an aggressive carcinoma driven by the BRD4-NUT fusion oncoprotein, which activates chromatin to promote expression of pro-growth genes. BET bromodomain inhibitors (BETi) impede BRD4-NUTs ability to activate genes and are thus a promising treatment but limited as monotherapy. The role of gene repression in NC is unknown. Here, we demonstrate that EZH2, which silences genes through establishment of repressive chromatin, is a dependency in NC. Inhibition of EZH2 with the clinical compound tazemetostat (taz) potently blocked growth of NC cells. Epigenetic and transcriptomic analysis revealed that taz reversed the EZH2-specific H3K27me3 silencing mark, and restored expression of multiple tumor suppressor genes while having no effect on key oncogenic BRD4- NUT-regulated genes. CDKN2A was identified as the only gene amongst all taz-derepressed genes to confer resistance to taz in a CRISPR-Cas9 screen. Combined EZH2 inhibition and BET inhibition synergized to downregulate cell proliferation genes resulting in more pronounced growth arrest and differentiation than either inhibitor alone. In pre-clinical models, combined taz and BETi synergistically blocked growth and prolonged survival of NC-xenografted mice, with all mice cured in one cohort. STATEMENT OF SIGNIFICANCEIdentification of EZH2 as a dependency in NC substantiates the reliance of NC tumor cells on epigenetic dysregulation of functionally opposite, yet highly complementary chromatin regulatory pathways to maintain NC growth. In particular, repression of CDKN2A expression by EZH2 provides a mechanistic rationale for combining EZH2i with BETi for the clinical treatment of NC.

cancer biology↗

bioRxiv2023

Immunomodulatory Leptin Receptor+ Sympathetic Perineurial Cells Protect Against Obesity by Facilitating Neuroendocrine-Mediated Brown Adipose Tissue Thermogenesis

Adipose tissues (ATs) are innervated by sympathetic nerves, which drive reduction of fat mass via lipolysis and thermogenesis. Here, we report a population of immunomodulatory leptin receptor (LepR)-expressing barrier cells which ensheath sympathetic axon bundles in adipose tissues. These LepR-expressing Sympathetic Perineurial Cells (SPCs) produce IL33, a factor for maintenance and recruitment of regulatory T cell (Treg) and eosinophils in AT. Brown adipose tissues (BAT) of mice lacking IL33 in SPCs (SPCIL33cKO) have fewer Treg and eosinophils, resulting in increased BAT inflammation. SPCIL33cKO mice are more susceptible to diet-induced obesity, independently of food intake. Furthermore, SPCIL33cKO mice have impaired adaptive thermogenesis, and are unresponsive to leptin-induced rescue of metabolic adaptation. We, therefore, identify LepR-expressing SPCs as a source of IL33 which orchestrate an anti-inflammatory environment in BAT, preserving sympathetic-mediated thermogenesis and body weight homeostasis. LepR+ IL33+ SPCs provide a cellular link between leptin and immune regulation of body weight, unifying neuroendocrinology and immunometabolism as previously disconnected fields of obesity research. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/539963v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@195fa69org.highwire.dtl.DTLVardef@16b2e5eorg.highwire.dtl.DTLVardef@1b12b62org.highwire.dtl.DTLVardef@973c1b_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- Sympathetic Perineurial Cells (SPCs) co-express LepR+ and IL33 - SPC-derived IL33 prevents BAT inflammation via Treg and eosinophil recruitment - Obesity is worsened in high fat diet-fed SPCIL33cKO mice, despite normal food intake - Adaptive thermogenesis is impaired in SPCIL33cKO mice - Rescue of metabolic adaptation to fasting by leptin is impaired in SPCIL33cKO mice - SPCs link leptin to immunometabolic regulation of body weight homeostasis

physiology↗

bioRxiv2022

SEC62 and TMX4 control asymmetric autophagy of the nuclear envelope upon LINC complex disassembly

The endoplasmic reticulum (ER) is a dynamic organelle of nucleated cells that produces proteins, lipids and oligosaccharides. The volume and the activities of the ER are adapted to cellular needs. They are increased upon induction of unfolded protein responses (UPR 1) and are reduced upon activation of ER-phagy programs 2. A specialized domain of the ER, the nuclear envelope (NE), protects the cell genome with two juxtaposed lipid bilayers, the inner and outer nuclear membranes (INM and ONM). SUN proteins in the INM form disulfide-bonded Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes with NESPRIN proteins in the ONM. These complexes set and maintain the width of the periplasmic space (PS), a continuum of the ER lumen, below the 50 nm 3-5 and in yeast prevent transmission of ER volume variations to the PS 6,7. Here we report that expansion of the mammalian ER upon homeostatic perturbations is transmitted to the NE, where the ONM forms large bulges. The process is reverted on recovery of ER homeostasis, by asymmetric vesiculation and autophagic clearance of ONM portions. Remodeling of the mammalian NE requires TMX4-driven reduction of the intermolecular disulfide bond stabilizing LINC complexes, the LC3 lipidation machinery, and the autophagy receptor SEC62, identified here as the first mammalian nucleo-phagy receptor.

cell biology↗

bioRxiv2019

Salivary gland macrophages assist tissue-resident CD8+ T cell immune surveillance

Tissue macrophages and tissue resident memory CD8+ T cells (TRM) play important roles for pathogen sensing and rapid protection of barrier tissues. To date, it is incompletely understood how these two cell types cooperate for efficient organ surveillance during homeostasis. Here, we used intravital imaging to show that TRM dynamically crawled along tissue macrophages in murine submandibular salivary glands (SMG) during the memory phase following a viral infection. Ex vivo confined SMG TRM integrated an unexpectedly wide range of migration modes: in addition to chemokine-and adhesion receptor-driven motility, SMG TRM displayed a remarkable capacity of autonomous motility in the absence of chemoattractants and adhesive ligands. This unique intrinsic SMG TRM motility was transmitted by friction and adaptation to microenvironmental topography through protrusion insertion into permissive gaps. Analysis of extracellular space in SMG using super-resolution shadow imaging showed discontinuous attachment of tissue macrophages to neighboring epithelial cells, offering paths of least resistance for patrolling TRM. Upon tissue macrophage depletion, intraepithelial SMG TRM showed decreased motility and reduced epithelial crossing events, and failed to cluster in response to local inflammatory chemokine stimuli. In sum, our data uncover a continuum of SMG TRM migration modes and identify a new accessory function of tissue macrophages to facilitate TRM patrolling of the complex exocrine gland architecture.\n\nOne sentence summaryCombined in vitro and in vivo imaging of salivary gland-resident tissue memory CD8+ T cells (TRM) uncovers their unique migratory behavior and describes a novel accessory function of tissue macrophages to assist TRM surveillance.

immunology↗