Biology subjects

Valitutti, S.

Publications and source records attributed to Valitutti, S..

10 recordsLinked to original sources

bioRxiv2026

Sustained antigenic engagement programs multidimensional CTL states with distinct cytotoxic capacities

Cytotoxic T lymphocytes (CTLs) eliminate infected or malignant cells through repeated target-cell encounters, yet how they sustain cytotoxic activity during extended antigenic stimulation remains poorly understood. In particular, it remains unclear whether preserved population-level killing reflects maintenance of a uniform effector phenotype or diversification into functionally distinct CTL populations. Here, we combined live-cell imaging, high-dimensional cytometry, single-cell RNA sequencing, phenotypic trajectory analysis, population sorting, and rechallenge killing assays to define how human clonal CTLs remodel during sustained antigenic engagement. Prior antigen encounter altered the kinetics and pattern of target-cell membrane permeabilization but did not compromise overall subsequent killing upon rechallenge. Single-cell transcriptomic profiling after prolonged engagement revealed broad remodeling of antigen-experienced CTLs and heterogeneous cytotoxic transcriptional programs. Time-resolved scRNA-seq further showed that these functional dimensions remodeled with distinct kinetics, with rapid induction of activation- and cytotoxicity-associated programs, later remodeling of vesicular-trafficking programs, and progressive reduction of adhesion/ contact interaction -associated programs. High-dimensional cytometry showed that this temporal remodeling was accompanied by progressive redistribution of CTLs across a multidimensional protein-expression landscape defined by distinct combinations of cytotoxic, vesicular/degranulation, activation, cytokine, adhesion, and regulatory features. Phenotypic trajectory analysis organized this protein landscape along a temporal remodeling axis, with early lytic-granule-rich profiles giving way to multiple late-engaged phenotypes with distinct combinations of functional protein features. Surface-marker-defined late CTL populations displayed distinct killing capacities upon rechallenge, demonstrating that preserved population-level cytotoxicity can coexist with substantial functional heterogeneity. Finally, CAR T cells exposed to CD19+ targets retained cytotoxic activity upon rechallenge and acquired multidimensional phenotypic features that partially overlapped with the remodeling framework identified in clonal CTLs. Reanalysis of longitudinal patient-derived CAR T-cell transcriptomes further showed that related functional programs were dynamically organized after infusion, with the features distinguishing responder and non-responder tisa-cel samples changing between the infusion product and day 7. Together, these findings indicate that sustained CTL killing is accompanied by progressive multidimensional remodeling and diversification rather than maintenance of a fixed effector phenotype. Our findings support a model in which population-level cytotoxic activity can be maintained while the CTLs composing that population progressively diversify into functionally distinct states during sustained antigenic engagement.

immunology↗

bioRxiv2026

Selective granzyme B replenishment dominates the molecular switch for sustained cytotoxicity in effector CD8+ T lymphocytes

Cytotoxic T lymphocytes (CTLs) are highly efficient multiple killer cells, yet how they sustain killing after the initial discharge of canonical lytic granules remains incompletely understood. Late-phase cytotoxicity is often attributed to death receptor signaling or cytokine-mediated mechanisms, but whether CTLs also remodel their intrinsic cytotoxic machinery during prolonged engagement remains unclear. Here, we show that human CTLs maintain cytotoxic resilience through selective remodeling of their lytic program, marked by preferential preservation of granzyme B (GzmB). Following acute degranulation and reduction of mature perforin, CTLs selectively replenish GzmB through de novo protein synthesis, whereas perforin and several other granule-associated components show limited restoration. Replenished GzmB becomes redistributed into vesicular compartments with reduced overlap with mature perforin and is delivered to successive targets during sustained cytotoxic responses. Under conditions of limited detectable mature perforin, CTLs continue to deliver enzymatically active GzmB, induce altered calcium responses in target cells, and support productive but kinetically heterogeneous killing. This GzmB-preserved effector state is maintained and further accentuated during engagement with resistant melanoma cells. Together, our findings identify selective GzmB renewal as a central feature of sustained CTL cytotoxicity and reveal an adaptive remodeling of the cytotoxic machinery that may support CTL resilience during prolonged immune responses.

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↗

bioRxiv2025

Isolation of functional supramolecular attack particles (SMAPs)

High density cultures of the GMP compatible NK-92 natural killer cell line release heterogeneous extracellular particles (EP), notably extracellular vesicles (EV) and non-vesicular extracellular particles (NVEP). The NVEP include a small proportion of supramolecular attack particles (SMAPs), the direct cytotoxic potential of which suggests therapeutic promise, yet scalable enrichment and functional evaluation alongside other extracellular particles (EPs) have been lacking. Here, we develop a high-resolution serial size-exclusion liquid chromatography (SELC) workflow that resolves EPs into 4 fractions F1-F4, in which EV are enriched in F2 and SMAPs are enriched in F3. Multi-modal characterization using Nanoparticle Tracking Analysis (NTA), Nano Flow Cytometry (Nano FCM), Transmission Electron Microscopy (TEM), Total Internal Reflection Microscopy (TIRFM) and Proteomics) demonstrated that F2 NK-92 EVs are [~]130-150 nm particles enriched for complement proteins, whereas SMAPs (F3) are 100-110 nm particles enriched for cytotoxic proteins (PRF1, GZMB) and SMAP shell components (e.g., THBS1, THBS4). Functionally, Ca2+-stabilized SMAPs (F3) trigger caspase-3-dependent apoptosis in tumor cell lines with robust dose responses, while F2 lacks direct cytotoxicity in vitro. Ex vivo, F3 SMAPs kill patient-derived breast cancer and chronic lymphocytic leukaemia cells (CLL) in a concentration-dependent manner. In NSG mice, intra-tumoral treatment with SMAPs (F3) restrains the growth of aggressive B16F10 melanoma and PANC-1 pancreatic cancer, confirming in vivo functionality. These results establish a scalable method to isolate and compare NK-derived particle classes and provide a foundation for targeted SMAP engineering as a promising approach for treatment of solid tumours. Significance StatementCell therapies face barriers in solid tumours, including lack of entry and suppression. NK cells release extracellular particles (EPs), including supramolecular attack particles (SMAPs), that can traffic into tumour tissue. We define a practical, scalable chromatography process that resolves NK-derived SMAPs from other EPs and demonstrate that SMAPs induce caspase mediated apoptosis in vitro and engage in cytotoxic lymphocyte independent anti-tumour activity in vivo. These insights open a route to engineering and testing SMAP-based therapeutics to overcome limitations of whole-cell approaches in solid cancers.

immunology↗

bioRxiv2025

Stenocytosis: a mechanism for supramolecular attack particle transfer at the CTL lytic synapse

Supramolecular attack particles (SMAPs) are recently discovered key components of the cytotoxic T lymphocytes (CTLs) lytic arsenal exhibiting autonomous cytotoxic behavior. Yet, how these particles might serve as synaptic weapons transferred from CTL into target cells within dynamic lytic synapses remains to be elucidated. In CTL interacting with immobilized stimuli, total internal reflection fluorescence microscopy (TIRFM) and rapid live 3D cell imaging showed that granules containing SMAPs navigate through narrow cortical actin cytoskeleton depletion areas to reach plasma membrane secretion hot-spots where SMAPs are released. In CTLs interacting with cognate target cells, correlative light-electron microscopy (CLEM) and structured illumination 3D imaging showed SMAP release into the synaptic cleft and penetration into the target cell trough an equally narrow gap in the target cell cortical actin cytoskeleton mirroring that formed in the CTL cytoskeleton, and just large enough to admit the SMAP into an early endosome. Our results reveal a previously undescribed process, stenocytosis (from the ancient Greek {sigma}{tau}{varepsilon}{nu}o{varsigma}, "narrow"), which allows hundred-nanometer-scale lytic particles to exit CTLs and enter target cells while evading synaptic defenses.

immunology↗

bioRxiv2024

Highly adaptable deep-learning platform for automated detection and analysis of vesicle exocytosis

Vesicle exocytosis is a fundamental component of intercellular communication, in all organisms. It has been studied for decades, using various imaging tools. Nevertheless, exocytosis research is still limited by the lack of reliable automated analysis procedures. To address this, we developed the Intelligent Vesicle Exocytosis Analysis Platform (IVEA), a nearly universal solution for analyzing exocytosis acquired with live cell imaging. IVEA is applicable to a wide variety of experimental model systems, microscopes and reporter fluorophores. IVEA combines state-of-the-art deep-learning and computer vision regimes to enable fully automated analysis of large data. IVEA runs as a FIJI plugin and does not require prior training or human intervention. IVEA is 60 times faster than manual analysis and is able to detect rare events often missed by the human eye. Overall, IVEA represents a breakthrough in the analysis of cellular secretory mechanisms and has a transformative potential for the exocytosis imaging field.

bioengineering↗

bioRxiv2024

Pore formation at the lytic synapse triggers the canonical pyroptotic cell death pathway

Prokaryotic pore-forming toxins drive inflammasome activation and pyroptosis through K+-dependent activation of the canonical NLRP3/caspase-1/gasdermin D signaling axis. In this study, we hypothesized that perforin, a eukaryotic pore-forming protein released into the lytic synapse by antigen-specific cytotoxic T lymphocytes (CTLs) upon cognate antigen recognition, mimics the pro-pyroptotic activity of ancestral pore-forming toxins, complementing its role as a conduit for granzymes. Utilizing imaging and molecular approaches, we demonstrate that perforation of target cells upon CTL attack elicits swift K+ efflux followed by NLRP3-dependent activation of proinflammatory caspase-1 and its major substrate, the pyroptotic executioner gasdermin D (GSDMD). Acute target cell death upon CTL attack is gasdermin-dependent and demonstrates morphological and molecular features of pyroptosis, including pyroptotic body formation, cell bloating, plasma membrane rupture, and release of intracellular contents. Perforation of target cells by soluble perforin is sufficient to trigger rapid K+ efflux, caspase-1 activation, and pyroptosis. By contrast, sustained interaction with CTLs unmasks a delayed apoptotic phenotype in the remaining target cells. Interestingly, exposure of target cells to exogenous supramolecular attack particles (SMAPs) recapitulates this apoptotic phenotype, suggesting that soluble perforin and SMAPs play dichotomous roles in target cell death. Our results reveal a novel mechanism for engagement of pyroptotic machinery upon CTL attack, in which perforin itself can autonomously engage programmed cell death (PCD), highlighting the complexity and diversity of the CTL lytic arsenal.

immunology↗

bioRxiv2023

ISiCell: involving biologists in the design process of agent-based models in cell biology

Agent-based models are commonly used in biology to study tissue-scale phenomena by reproducing the individual behavior of the cells. They offer the possibility to study cellular biology at the individual cell scale to explore the basic behavior of cells which are responsible of the emergence of more complex phenomena at the tissue scale. Additionally, they can produce a predictive tool that will help taking decisions for biologic experiments based on in silico simulations. However these models require a good intercomprehension between the biologists and the modelers and thus it may take weeks or months to end up providing a usable prototype. To address this limitation, we propose a new methodology to facilitate the dialog between biologists and modelers and improve biologists involvement in the design of the model. For this purpose, UML diagrams, in particular, state-transition and activity diagrams, are used. They allow a better comprehension of the model for the biologists and offer a general frame for structuring models. Visualization of simulations is also used to have qualitative feedbacks from the biologist on the model. They are instrumental to validate or refine the prototype before exploring it. Alongside this methodology, we propose a web platform that enables to build state-transition and activity diagrams to describe a model and translate them into code. The generated code is then compiled on-the-fly and simulations are ready to visualize and explore. The platform also disposes of tools to directly visualize and manually explore the model. These tools allow for qualitative validation of the model and additional interaction with the biologists. Finally in this article, we show the capacity of our platform to reproduce models from the literature and to build new models starting from workshops with biologists. Its range of application is wide and includes immunology, oncology or cell biology. Author summaryWe developed a methodology based on diagrams to facilitate the dialog between computer scientists and biologists when building in silico models. The main idea is to limit misunderstandings and improve the involvement of the biologists in the prototyping process. For this purpose, we use visual methods to simplify the modeling phase. Alongside this methodology, we propose a web platform, called ISiCell, which enables to visually code thanks to diagrams that will be translated into code. The platform allows for compiling the generated code on the fly and to visualize and explore the model directly with the platform. The strong advantage of the platform is that one day workshop biologist/modeler allows to build new models. Additionally, we were able to reproduce models from the literature within the modeling platform showing the versatility of the tool. Our long-term objective is to use our methodology and platform in new contexts to develop new models. We intend the make the platform more user friendly in order to expand the community of users. Involving biologists in the conception of in silico models might improve their acceptability in the community.

systems biology↗

bioRxiv2021

T helper cell-licensed mast cells promote inflammatory Th17 cells

CD4+ T helper cells (Th) infiltrate sites of inflammation and orchestrate the immune response by instructing local leukocytes. Mast cells (MCs) are tissue sentinel cells particularly abundant in skin and mucosa. Here, we analyzed the interplay between human MCs and Th cells and, through the application of RNAseq and functional assays, showed that Th cells induced a specific transcriptomic program in helped MCs (named here MCTH) driving them toward an inflammatory phenotype. The gene signature of MCTH indicated that MCs helped by Th cell acquired in turn the capacity to regulate effector T cell response through wide-range of soluble and membrane ligands. Accordingly, we showed that MCTH promoted Th17 cells and notably an inflammatory subset of Th17, producing both IFN-{gamma} and GM-CSF, through a PGE2 and IL-1{beta} axis. Our findings demonstrate that activated effector/memory CD4+ T cells activate and instruct resting MCs toward a specific differentiated pro-inflammatory phenotype endowed with the capacity to speak back to effector T cells and to mold their functions.

immunology↗

bioRxiv2020

Stochastic asymmetric repartition of lytic machinery in dividing human CD8+ T cells generates heterogeneous killing behavior

Cytotoxic immune cells are endowed with a high degree of heterogeneity in their lytic function, but how this heterogeneity is generated is still an open question. We therefore investigated if human CD8+ T cells could segregate their lytic components during telophase, using imaging flow cytometry, confocal microscopy and live cell imaging. We show that CD107a+-intracellular vesicles, perforin and granzyme B unevenly segregate in a constant fraction of telophasic cells during each division round. Mathematical modeling posits that unequal lytic molecule inheritance by daughter cells results from the random distribution of lytic granules on the two sides of the cleavage furrow. Finally, we establish that the level of lytic compartment in individual CTL dictates CTL killing capacity. Together, our results show the stochastic asymmetric distribution of effector molecules in dividing CD8+ T cells. They propose uneven mitotic repartition of pre-packaged lytic components as a mechanism generating non-hereditary functional heterogeneity in CTL.

immunology↗