Biology subjects

Lemaitre, F.

Publications and source records attributed to Lemaitre, F..

8 recordsLinked to original sources

bioRxiv2026

T cell engagers convert acute tumor killing into endogenous vaccination

T cell engagers (TCEs) redirect T cells to kill tumors by cross-linking CD3{varepsilon} to a tumor antigen, independently of T cell receptor specificity. Some patients achieve remissions that persist long after drug clearance, but whether TCEs can couple acute cytotoxicity to the generation of lasting adaptive immunity remains unknown. Here, using an immunocompetent mouse model for clinical-grade human TCEs, we show that TCE-mediated killing initiates an endogenous vaccination cascade. Intravital two-photon imaging reveals a perforin-dependent cytotoxic wave that peaks within 24 hours, then subsides despite massive T cell accumulation. Single-cell transcriptomics and adoptive transfers demonstrate that memory CD8 T cells, the principal effector reservoir in mouse and human, transit through a progenitor-like state before differentiating into cytotoxic effectors with recall competence. Crucially, tumor lysis fuels cross-priming of tumor-specific CD8 T cells by XCR1 type 1 conventional dendritic cells, a cDC1-dependent step essential for durable tumor control and protective memory upon rechallenge.

immunology↗

bioRxiv2026

KLRG1 identifies circulating cytotoxic CD4 T cells with selective anti-tumor function in human cancer

Given the critical role of CD4 T cells in anti-tumor immunity, strategies to harness these cells for cancer immunotherapy are gaining increasing interest. Historically overshadowed by CD8 T cells, cytotoxic CD4 T cells can directly kill MHC class II-expressing tumor cells. However, the defining molecular signature and the mechanisms underlying their cytolytic activity remain poorly understood, particularly in cancer patients. Here, using ex vivo single-cell transcriptomic and spatial analyses of CD4 T cells from paired blood and tumor samples of melanoma patients, we identified Killer Cell Lectin-Like Receptor G1 (KLRG1) as a defining surface marker of cytotoxic CD4 T cells. The CD4+ KLRG1+ T cell subset was notably enriched among circulating cells compared with tumor-infiltrating populations, which were instead enriched in T follicular helper (Tfh) states. Functionally, KLRG1+ CD4 T cells expressed elevated levels of cytotoxic genes and exhibited superior tumor-killing capacity compared with their KLRG1- counterparts. We demonstrated that their cytotoxicity is granulysin-dependent, as confirmed by CRISPR/Cas9-mediated gene deletion. Mechanistically, CD4 T cells spared MHC class II+ cells lacking the KLRG1 ligands CD324 and CD325, such as professional antigen-presenting cells (APCs), indicating that cytotoxicity was selectively directed towards tumor cells while preserving immune cells. Finally, by investigating how the tumor microenvironment may impair CD4 T cell cytotoxicity, we showed that tumor-derived factors, including interleukin-6 (IL-6), are key drivers promoting the transition of cytotoxic CD4 T cells toward a Tfh phenotype. In summary, our findings define KLRG1 as a defining cell surface marker of cytotoxic CD4 T cells in cancer patients, as well as a key regulator that protects MHC class II+ APCs. Moreover, targeting the IL-6 signalling pathway may enhance CD4 T cell anti-tumor cytotoxicity, offering new avenues for cancer immunotherapy.

immunology↗

bioRxiv2026

CAR-MACROPHAGES ACTIVATE ANTI TUMOR T CELLS IN THE ABSENCE OF PHAGOCYTOSIS

Macrophages are highly abundant within the tumor microenvironment and serve as an essential bridge between innate and adaptive immune responses. Thus, they have emerged as promising candidates for chimeric antigen receptor (CAR)-based therapeutic strategies. Previous studies demonstrated that adenovirally-transduced CAR-macrophages (CAR-M), used in clinical trials, can perform tumor cell phagocytosis, reshape the tumor microenvironment towards a proinflammatory state, and promote host T cell activation. However, how early interactions between CAR-M and tumor cells shape subsequent T cell effector functions remains poorly understood. Particularly, uncoupling the adenoviral-induced proinflammatory phenotype from CAR-M effector functions remains to be dissected. Here, using phagocytosis-optimized CAR-Ms, we demonstrated that T cells are crucial mediators of CAR-M therapeutic efficacy. Mechanistically, we identify that CAR-M-derived cytokines and chemokines are key drivers of T cell functions while antigen cross presentation appears largely dispensable. Dynamic imaging of CAR-M-tumor cell interactions revealed heterogenous phagocytic abilities, regardless of contact duration. Finally, we show that non-phagocytic interactions can instruct pro-inflammatory macrophage repolarization and subsequent T cell activation. Altogether, these findings define an alternative mode of CAR-M action in which CAR engagement alone, independently of target cell uptake, is sufficient to enhance T cell functions. Our study underscores the importance of non-phagocytic CAR signaling as an additional mechanism for CAR-M effector functions opening new avenues for the design of next generation CAR for macrophage-based therapies.

immunology↗

bioRxiv2025

HAK-actin, U-ExM-compatible probe to image the actin cytoskeleton

Expansion microscopy (ExM) enables super-resolution imaging by physically enlarging biological samples. While ExM has been successfully applied to study the intracellular microtubule cytoskeleton, reliable probes for visualizing actin fibers remain limited. Here, we present HAK-actin, an engineered actin probe compatible with post-labeling Ultrastructure Expansion Microscopy (U-ExM). We show that HAK-actin delivers robust and uniform actin staining across diverse systems, including human cells, microbial eukaryotes, and mouse retinal tissue. This tool provides a simple, versatile, and reproducible solution for actin cytoskeleton visualization, addressing a critical need in cell biology.

cell biology↗

bioRxiv2025

Unveiling the Molecular Architecture of T Cells and Immune Synapses with Cryo-Expansion Microscopy

Cellular communication is critical for anti-cancer immunity, with tumor cell killing occurring at immunological synapses (IS) formed between effector immune cells and target tumor cells. While optical super-resolution microscopy (SRM) has enlightened the spatial organization of the IS mostly in regular immune cells, visualizing the nanoscale architectural features of IS in its native state, including 3D receptor distribution and the ultrastructural details of the lytic granule release remains challenging. Using cryo-expansion microscopy (cryo-ExM), we unravel the cellular architecture of activated T cells and T cell-target cell pairs. Our approach visualizes actin and microtubule networks during synapse formation, membrane topography, and the distribution of signaling molecules and lytic granules of different types, offering novel insights into IS organization. Finally, we apply U-ExM to glioblastoma tissue, visualizing T cells and their lytic content in situ, highlighting its potential for pre-clinical immunotherapy studies.

immunology↗

bioRxiv2025

Cell-free Reconstitution Reveals Synergistic Stabilization of Microtubule Doublets by PACRG and FAP20

Ciliary microtubule doublets, consisting of an incomplete B-tubule on the surface of a complete A-microtubule, form the fundamental structural framework of motile and sensory cilia/flagella. Ciliary proteins PACRG and FAP20, which are essential for flagellar stability and function, localize to the junction between the A-and the B-tubule. However, their precise role in microtubule doublet assembly and dynamics remains unknown. Here, using a cell-free assay, TIRF-microscopy, and cryo-electron tomography, we demonstrate that in combination PACRG and FAP20 stabilize B-tubule dynamics and microtubule doublet architecture. We show that together these proteins localize to the B-tubules in distinct, high-density patches, which locally stabilize B-tubule by decreasing its depolymerization velocity and increasing its rescue frequency. Cryo-tomography of in vitro reconstructed microtubule doublets in presence of PACRG and FAP20 reveals reduced B-tubule curvature fluctuations, promoting a more rigid and aligned conformation. Altogether, our findings suggest that PACRG and FAP20 function synergistically to reinforce microtubule doublet stability, ensuring ciliary integrity and function.

cell biology↗

bioRxiv2020

ALG-2 interacting protein-X (Alix) is required for activity-dependent bulk endocytosis at brain synapses

In chemical synapses undergoing high frequency stimulation, vesicle components can be retrieved from the plasma membrane via a clathrin-independent process called activity dependent bulk endocytosis (ADBE). Alix (ALG-2 interacting protein X)/ PDCD6IP) is an adaptor protein binding to ESCRT and endophilin-A proteins which is required for clathrin-independent endocytosis in fibroblasts. Alix is expressed in neurons and concentrates at synapses during epileptic seizures. Here, we used cultured neurons to show that Alix is recruited to presynapses where it interacts with, and concentrates endophilin-A during conditions triggering ADBE. Using Alix knockout (ko) neurons we showed that this recruitment, which requires interaction with the calcium-binding protein ALG-2, is necessary for ABDE. We also found that presynaptic compartments of Alix ko hippocampi display subtle morphological defects compatible with flawed synaptic activity and plasticity detected electrophysiologically. Furthermore, mice lacking Alix in the forebrain undergo less seizures during kainate-induced status epilepticus and reduced propagation of the epileptiform activity. These results thus show that impairment of ADBE due to the lack of neuronal Alix alters synaptic recovery during physiological or pathological repeated stimulations.

neuroscience↗

bioRxiv2020

Imaging the mechanisms of anti-CD20 therapy in vivo uncovers spatiotemporal bottlenecks in antibody-dependent phagocytosis

Anti-CD20 monoclonal antibody (mAb) represents an effective strategy for the treatment of B cell malignancies that may involve complement activity, antibody dependent cellular cytotoxicity (ADCC) and phagocytosis (ADP). While ADP mediated by Kupffer cells is essential to deplete circulating tumors, the relative contribution of each mechanism to the elimination of non-circulating targets has yet to be clarified. Using intravital imaging in a model of MYC-driven B cell lymphoma, we establish here the dominance and limitations of ADP in the bone marrow (BM). We found that tumor cells were stably residing in the BM with little evidence for recirculation. To quantify the contribution of different cytotoxic mechanisms in situ, we designed a dual fluorescent reporter to track phagocytosis and apoptosis in real-time. ADP by BM-associated macrophages was the primary mode of tumor elimination but was no longer active after one hour, resulting only in partial depletion. Moreover, macrophage density was strongly reduced in tumor-rich regions. Given their sessile phenotype, macrophages primarily targeted neighboring tumors, resulting in a substantial spatial constraint. Overcoming spatiotemporal bottlenecks in tumor-targeting Ab therapy represents a critical path towards the design of optimized therapies. Key pointsO_LIFunctional intravital imaging establishes antibody-dependent phagocytosis as the major mechanism acting at the tumor site during anti-CD20 therapy. C_LIO_LIA transient wave of phagocytosis and a limited macrophage density restrict the efficiency of anti-CD20 anti-tumor activity. C_LI

immunology↗