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Intranasal treatments, combined with vaccination, has the potential to slow mutational evolution of virusues by reducing transmission and replication. Here we illustrate the development of a SARS-CoV-2 receptor binding domain (RBD) nanoCLAMP and demonstrate its potential as an intranasally administered therapeutic. A multi-epitope nanoCLAMP was made by fusing a pM affinity single-domain nanoCLAMP (P2710) to alternate epitope binding nanoCLAMP, P2609. The resulting multimerised nanoCLAMP P2712 had sub-pM affinity for the Wuhan and South African (B.1.351) RBD (KD< 1 pM), and decreasing affinity for the Delta (B.1.617.2) and Omicron (B.1.1.529) variants (86 pM and 19.7 nM, respectively). P2712 potently inhibited ACE2:RBD interaction, suggesting its utility as a therapeutic. With an IC50= 0.4 ± 0.1 nM obtained from neutralization experiments using pseudoviral particles as well as patient cultured SARS-CoV-2 samples, nanoCLAMP P2712 protected K18-hACE2 mice from SARS-CoV-2 infection, reduced viral loads in the lungs and brains, and reduced associated upregulation of inflammatory cytokines and chemokines. Together, our findings warrant further investigation into the development of nanoCLAMPs as effective intranasally delivered COVID19 therapeutics.

The surface of the central nervous system (CNS) is protected by the meninges, which contain a dense network of meningeal macrophages (MMs). Here, we examined the role of tissue-resident MM in viral infection. MHC-II� MM were abundant neonatally, whereas MHC-II+ MM appeared over time. These barrier macrophages differentially responded to in vivo peripheral challenges such as LPS, SARS-CoV-2, and lymphocytic choriomeningitis virus (LCMV). Peripheral LCMV infection, which was asymptomatic, led to a transient infection and activation of the meninges. Mice lacking macrophages but conserving brain microglia, or mice bearing macrophage-specific deletion of Stat1 or Ifnar, exhibited extensive viral spread into the CNS. Transcranial pharmacological depletion strategies targeting MM locally resulted in several areas of the meninges becoming infected and fatal meningitis. Low numbers of MHC-II+ MM, which is seen upon LPS challenge or in neonates, corelated with higher viral load upon infection. Thus, MMs protect against viral infection and may present targets for therapeutic manipulation., Authors affiliations: (1) Centre d'Immunologie de Marseille-Luminy, Aix Marseille Université, Inserm, CNRS, Marseille, France (2) Ecole Normale Supérieure de Lyon, Université Claude Bernard – Lyon I, Université de Lyon, Lyon, France (3)Centre d'Immunophénomique, Aix Marseille Université , Inserm, CNRS, Marseille, France (4) TERI (Tumor Escape, Resistance and Immunity) Department, Centre de Recherche en Cancérologie de Lyon, Centre Léon Bérard, Université Claude Bernard – Lyon 1, Université de Lyon, Inserm, CNRS, Lyon, France (5) Viral Immunology and Intravital Imaging Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA (6) Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine, Shanghai, China (7) University Paris-Saclay, Inserm U1015, Gustave Roussy Cancer Center, Villejuif, France (8) Singapore Immunology Network, A*STAR, Singapore, Singapore (9) Centre for Inflammation Biology and Cancer Immunology, Cancer Research UK King's Health Partners Centre, School of Immunology and Microbial Sciences, King's College London, London SE1 1UL, UK (10) Lead contact

International audience; 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.

International audience; BackgroundThere is an urgent need of a new generation of vaccine that are able to enhance protection against SARS-CoV-2 and related variants of concern (VOC) and emerging coronaviruses.MethodsWe identified conserved T- and B-cell epitopes from Spike (S) and Nucleocapsid (N) highly homologous to 38 sarbecoviruses, including SARS-CoV-2 VOCs, to design a protein subunit vaccine targeting antigens to Dendritic Cells (DC) via CD40 surface receptor (CD40.CoV2).FindingsCD40.CoV2 immunization elicited high levels of cross-neutralizing antibodies against SARS-CoV-2, VOCs, and SARS-CoV-1 in K18-hACE2 transgenic mice, associated with viral control and survival after SARS-CoV-2 challenge. A direct comparison of CD40.CoV2 with the mRNA BNT162b2 vaccine showed that the two vaccines were equally immunogenic in mice. We demonstrated the potency of CD40.CoV2 to recall in vitro human multi-epitope, functional, and cytotoxic SARS-CoV-2 S- and N-specific T-cell responses that are unaffected by VOC mutations and cross-reactive with SARS-CoV-1 and, to a lesser extent, MERS epitopes.InterpretationWe report the immunogenicity and antiviral efficacy of the CD40.CoV2 vaccine in a preclinical model providing a framework for a pan-sarbecovirus vaccine.FundingsThis work was supported by INSERM and the Investissements d'Avenir program, Vaccine Research Institute (VRI), managed by the ANR and the CARE project funded from the Innovative Medicines Initiative 2 Joint Undertaking (JU).

Despite considerable progress in understanding the biology and genetics of breast cancer, the development of effective therapies needs physiological and predictive preclinical models. In this context, breast cancer (BC) patient-derived xenograft (PDX) models have become a standard tool as they reproduce the biology of tumors of origin, in term of histology, genotype and response to chemotherapy. They have proven their relevance in the study of pathways leading to the development and progression of cancer, to the mechanisms linked to tumor resistance and to the identification of novel effective therapies. We present a preclinical platform of over 60 fully characterized BC PDX models and their in vitro cell derivatives for preclinical evaluation of new treatment modalities. Our platform consists of a PDX collection of 43 TNBC, 6 ER+, 4 HER2+, 6 Luminal B models (ER+ HER2+) and 14 cellular models derived from these PDXs, representing the variety of BC. PDX models were obtained by transplantation of post-surgery tumor specimens either by grafting of tumor fragments in the interscapular region of nude mice or by injection of tumor cells into the fat pad of NOD-Scid mice. Molecular analyses were done included gene expression, gene copy number, whole exome sequencing and IHC markers staining. In vivo drug efficacy assays were performed with standards of care as single agent or in combinations. This PDX panel mostly reflects the molecular heterogeneity of breast cancer and reproduce accurately the molecular and drug response profile of human tumors. It provides an invaluable tool for translational research. It is widely used to performed standard drug evaluation but also “Mouse Clinical Trials” (MCT) in vivo screens to provide more predictive preclinical data on single-agent or combination drug efficacy. Engrafted on highly immunodeficient mice humanized with human PBMCs or CD34+ cells, these PDX models allow bispecific T-Cell engager antibody testing or immune-checkpoint inhibitors evaluation. In addition to these PDX panel, we derived cellular models (PDXDCs) to offer a time- and cost-effective preclinical screening tool. PDXDCs were obtained from dissociated PDX tumors cultured under different media and matrix conditions. They were characterized by comparison with the parental PDX by Short Tandem Repeat (STR) profiling before performing a master bank. WES and RNASeq molecular analyses were done and in vitro drug sensitivity was compared with their parental PDX in vivo drug response. Overall, the results show that this PDXDC panel reproduced in vitro the in vivo drug response profile of the original PDXs with various therapies. This BC PDX panel and in vitro cell derivatives provide a powerful preclinical platform to improve our knowledge on BC biology and to rapidly evaluate response to new treatments and translate this knowledge to the clinic. Citation Format: Delphine Nicolle, Aurore Gorce, Marie Tavernier, Elisabetta Marangoni, Didier Decaudin, Christophe Ginestier, Emmanuelle Charafe-Jaufret, Judith Passildas, Nina Robin, Robert Clarke, Erwan Corcuff, Anaïs Joachim, Bernard Malissen, Ana Zarubica, Hervé Luche, Jean-Gabriel Judde, Olivier Déas. A preclinical platform of breast cancer PDX and derived cellular models as a tool for pharmacological screening and functional studies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4677.

TNFRF-14/HVEM is the ligand for BTLA and CD160 negative immune co-signaling molecules as well as viral proteins. Its expression is dysregulated with an overexpression in tumors and a connection with tumors of adverse prognosis. We developed C57BL/6 mouse models co-expressing human huBTLA and huHVEM as well as antagonistic monoclonal antibodies (mAbs) that completely prevent the interactions of HVEM with its ligands. Here, we show that the anti-HVEM18-10 mAb increases primary human αβ-T cells activity alone (CIS-activity) or in the presence of HVEM-expressing lung or colorectal cancer cells in vitro (TRANS-activity). Anti-HVEM18-10 synergizes with anti-PD-L1 mAb to activate T cells in the presence of PDL-1 positive tumors, but is sufficient to trigger T cell activation in the presence of PD-L1 negative cells. In order to better understand HVEM18-10 effect in vivo and especially disentangle its CIS and TRANS effects, we developed a knock-in (KI) mouse model expressing human BTLA (huBTLA+/+) and a KI mouse model expressing both human BTLA and human HVEM (huBTLA+/+/huHVEM+/+ (DKI)). In vivo pre-clinical experiments performed in both mouse models showed that HVEM18-10 treatment was efficient to decrease human HVEM+ tumor growth. In the DKI model, anti-HVEM 18-10 treatment induces a decrease of exhausted CD8+ T cells and regulatory T cells and an increase of Effector memory CD4+ T cells within the tumor. Interestingly, mice which completely rejected tumors (± 20%) did not develop tumors upon re-challenge in both settings, therefore showing a marked T cell-memory phenotype effect. Altogether, our preclinical models validate anti-HVEM18-10 as a promising therapeutic antibody to use in clinics as a monotherapy or in combination with existing immunotherapies (anti-PD1/anti-PDL-1/anti-CTLA-4). Citation Format: Laurent GORVEL, Clemence Demerle, Marielle Mello, Sonia Pastor, Clara Degos, Ana Zarubica, Fabien Angelis, Frederic Fiore, Jacques Nunes, Bernard Malissen, Laurent Greillier, Geoffrey Guittard, Herve Luche, Fabrice Barlesi, Daniel Olive. Anti-HVEM mAb therapy improves antitumoral immunity both in vitro and in vivo, in a novel transgenic mouse model expressing human HVEM and BTLA molecules challenged with HVEM expressing tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5184.

Identification of major cellular players involved in progression of cancer is key to success of new immunotherapies. It is however a daunting challenge due to the complex interplay between tumor cells and the immune system. Component of innate and adaptive immunity may be directed to a pro- or anti-tumor function. In order to cope with the complexity of the tumor microenvironment (TME), it is necessary to use an experimental approach characterizing the heterogeneity of cell types, the evolution of their relative proportion and their fine-tuned functional specificity. This approach should lead to the identification of new cellular biomarkers characteristics of different stages of tumor cancer progression. To decipher the impact of immunotherapy treatments, cellular phenotyping of leukocytes infiltrating TME but also those present in peripheral organs is necessary. In order to increase our understanding in the precise mode of action of anti-PD1 treatment at the cellular level in sensitive and unsensitive syngeneic models, we investigated immunophenotypes and responses to immune checkpoint inhibitor (ICI) of several hallmark tumor models (MC38, CT26, B16F10, B16-OVA, RENCA, EMT6) in immunocompetent mouse models by flow and mass cytometry. We compared growth kinetics and profiled the immune cell composition of tumor microenvironment (TME), draining lymph node (dLN) and blood in order to establish immune-phenotypic cell signatures that correlates with treatment efficacy. Supervised, unsupervised and integrative data analysis arewere used to identify significant changes across different experimental settings. Our results indicate that each model possesses a unique tumor-immune infiltrate profile that can be modulated with immunotherapies. Overall, these studies provide an important resource of highly well-characterized syngeneic tumor model and highlight the importance of tumor immune landscape changes across models that will drive selection of the most appropriate model to test novel immunotherapeutic agents and enhance our translation of knowledge from syngeneic models to human tumors. Citation Format: Anais Joachim, Emilie Maturin, Marielle Mello, Lillia Hadjem, Magali Grange, Olivier Deas, Ana Zarubica, Bernard Malissen, Hervé Luche. Deep Immuno-profiling of syngeneic tumor mouse models for preclinical studies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4630.

Lung cancer remains the first cause of cancer-related deaths worldwide, of which Non-Small Cell Lung Cancer (NSCLC) represents more than 80% of patients with advanced disease at the time of diagnosis. NSCLC is a highly heterogenous disease, and the identification of its main actionable oncogenic drivers (i.e. EGFR, ALK, PI3K/AKT/mTOR, RET, MET, BRAF and NTRK/ROS1) and the development of specific inhibitors against these targets has transformed therapeutic care. In addition, immune-checkpoint therapy has emerged as an indispensable treatment modality, especially for patients lacking actionable oncogenic drivers, although biomarkers for predicting response to immune-checkpoint inhibition have remained elusive. Despite these new therapeutic options, NSCLC remains a lethal disease in the majority of patients due to tumor plasticity and selection leading to frequent resistance development and disease progression. Efforts are therefore needed to identify drugs and drug combinations that can prevent or overcome these resistance pathways. Patient-Derived Xenografts (PDX) models developed in immune-compromised mice recapitulate the disease more faithfully than any other in vivo model in terms of histopathologic and genomic features. They have proven their relevance in the study of pathways leading to the development and progression of cancer, to the mechanisms linked to tumor resistance and to the identification of novel effective therapies, facilitating the translation of preclinical results in the clinical setting. We describe a platform of over 35 NSCLC PDX models covering most of the molecular diversity of the disease, that have been fully characterized at the molecular level and for their response to a panel of cytotoxic chemotherapies and targeted therapies. These NSCLC PDX models have been established in immune-deficient mice from tumor biopsies collected in treatment-naïve patients or in patients having acquired resistance following an initial objective response to a variety of targeted inhibitors (EGFRi, ALKi, ROSi, BRAFi,⋯) in the MATCH-R clinical trial. In addition, 4 PDX models were established from circulating tumor cells (CTC) isolated from the blood of advanced NSCLC patients. Finally, some of these NSCLC PDX models were established in highly immunodeficient mice humanized with human PBMCs or CD34+ cells allowing testing of efficacy of bispecific T-Cell engager antibody or immune-checkpoint inhibitors. This panel of NSCLC PDX models provides a powerful preclinical platform to improve our knowledge on the mechanisms underlying resistance to treatment and to rapidly evaluate response to new treatments and translate this knowledge to the clinic. Citation Format: Delphine Nicolle, Laura Brulle-Soumare, Katell Mevel, Ludovic Bigot, Tala Tayoun, Benjamin Besse, Françoise Farace, Luc Friboulet, Didier Decaudin, Erwan Corcuff, Anaïs Joachim, Bernard Malissen, Ana Zarubica, Hervé Luche, Jean-Gabriel Judde, Olivier Deas. Characterization of a PDX panel covering molecular diversity of non-small cell lung cancer to accelerate the development of precision therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4678.

Preclinical tumor oncology research relies historically on the analysis of mouse or human tumor cell lines implanted onto severely immune-deficient mouse models carrying nude, SCID or Rag mutations on different genetical background (C57Bl/6N, BalbC, NMRI, CB17 or NOD). These lines harbor defects in several leukocyte lineages among which Tc, Bc and NKc might be affected. Humanized mice are emerging models that have been transplanted with human cells or tissues (and/or equipped with human transgenes). Currently, the most advanced strains are the nonobese diabetic, severe combined immunodeficiency (NOD-SCID) mouse with complete disruptions in the interleukin-2 (IL-2) common γ-chain (IL2R γ null) receptor (NSG) and BALB/c Rag2−/− IL2R γ null SirpaNOD mice (BRGS).1 Juvenile chimera NSG or BRGS are reconstituted with hematopoietic progenitor cell (HPCs) from human cord blood yielding robust engraftment of a human immune system (HIS).. Recent work showing the binding of human IgG4 to mouse FcgR receptor reducing clinical efficacy of therapeutic antibodies highlights the need to characterize extensively the residual immune system present in these models prior humanization in order to precisely evaluate immunotherapies. In other to fulfill this task, we genetically modified the mouse genome introducing mutations involved in Tc, Bc and NKc development. We then immuno-phenotyped over 20 lines of mouse mutants produced from the same animal house to minimize the impact of microbiota on innate immune cell populations. High content cytometry analysis of BM, spleen, thymus and peripheral blood was performed in a standardized manner. The resource obtained of these immunotypes was used to highlight similarities and differences among these lines across several genetical background. This work has shown instrumental in selecting the most appropriate model to use for humanization followed before tumor implantation. Citation Format: Hervé Luche, Lillia Hadjem, Marielle Mello, Priscilla Canavese, Fabien Angelis, Anais Joachim, Sylvie Bouilly, Frederic Guinut, Frederic Fiore, Bernard Malissen, Ana Zarubica, Erwan Corcuff. Systematic immune-profiling of immune-deficient mouse models: A rational to select ideal host for tumor implantation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5190.

SummaryLung-resident memory B cells (MBCs) provide localized protection against reinfection in the 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. scRNA-seq analysis and confocal imaging revealed that two main transcriptionally distinct subsets of MBCs colonize the lung peribronchial niche after infection. These subsets arise from different progenitors and are both class-switched, somatically mutated and intrinsically biased in their differentiation fate towards plasma cells. Combined analysis of antigen-specificity and B cell receptor repertoire unveiled a highly permissive selection process that segregates these subsets into “bona fide” virus-specific MBCs and “bystander” MBCs with no apparent specificity for eliciting viruses. 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.

COVID-19 is the biggest pandemic the world has seen this century. Alongside the respiratory damage observed in patients with severe forms of the disease, gastrointestinal symptoms have been frequently reported. These symptoms (e.g., diarrhoea), sometimes precede the development of respiratory tract illnesses, as if the digestive tract was a major target during early SARS-CoV-2 dissemination. We hypothesize that in patients carrying intestinal SARS-CoV-2, the virus may trigger epithelial barrier damage through the disruption of E-cadherin (E-cad) adherens junctions, thereby contributing to the overall gastrointestinal symptoms of COVID-19. Here, we use an intestinal Caco-2 cell line of human origin which expresses the viral receptor/co-receptor as well as the membrane anchored cell surface adhesion protein E-cad to investigate the expression of E-cad after exposure to SARS-CoV-2. We found that the expression of

Improving reproducibility and replicability in preclinical research is a widely discussed and pertinent topic, especially regarding ethical responsibility in animal research. INFRAFRONTIER, the European Research Infrastructure for the generation, phenotyping, archiving, and distribution of model mammalian genomes, is addressing this issue by developing internal quality principles for its different service areas, that provides a quality framework for its operational activities. This article introduces the INFRAFRONTIER Quality Principles in Systemic Phenotyping of genetically altered mouse models. A total of 11 key principles are included, ranging from general requirements for compliance with guidelines on animal testing, to the need for well-trained personnel and more specific standards such as the exchange of reference lines. Recently established requirements such as the provision of FAIR (Findable, Accessible, Interoperable, Reusable) data are also addressed. For each quality principle, we have outlined the specific context, requirements, further recommendations, and key references.

Macrophages are central players in both lipid metabolism and innate immunity. Their determinant role in the pathogenesis of atherosclerosis is under the control of the ATP-binding cassette transporter (ABCA1), which by minimizing cellular lipid content, limits development of pro-inflammatory foam cells. Considering the differential contribution of monocyte subsets to the generation of vascular lesions we analyzed the immunophenotype of ABCA1-expressing cells in the myeloid lineage, by the combined use of flow cytometry and real-time quantitative RT-PCR. ABCA1 expression is limited to "non-inflammatory" Ly6C(lo) circulating monocytes and tissue-resident macrophages expressing markers of alternative activation. In ABCA1(-/-) peritoneal macrophages the transcriptional programs induced by LPS/IFN-gamma or IL-4 cytokines are altered and deviated phosphorylation patterns of STAT transcriptional regulators in response to stimuli are observed.

The ABCA1 transporter is the prototype of the A class of mammalian adenosine triphosphate binding cassette transporters and one of the largest members of this family. ABCA1 has been originally identified as an engulfment receptor on macrophages and, more recently, it has been shown to play an essential role in the handling of cellular lipids. Indeed by promoting the effluxes of membrane phospholipids and cholesterol to lipid-poor apoprotein acceptors, ABCA1 controls the formation of high-density lipoproteins and thus the whole process of reverse cholesterol transport. A number of additional phenotypes have been found in the mouse model of invalidation of the ABCA1 gene. In spite of their clinical diversity, they all are extremely sensitive to variations in the physicochemical properties of the cell membrane, which ABCA1 controls as a lipid translocator.

International audience; ABCA1 belongs to the A class of ABC transporter, which is absent in yeast. ABCA1 elicits lipid translocation at the plasma membrane through yet elusive processes. We successfully expressed the mouse Abca1 gene in Saccharomyces cerevisiae. The cloned ABCA1 distributed at the yeast plasma membrane in stable discrete domains that we name MCA (membrane cluster containing ABCA1) and that do not overlap with the previously identified punctate structures MCC (membrane cluster containing Can1p) and MCP (membrane cluster containing Pma1p). By comparison with a nonfunctional mutant, we demonstrated that ABCA1 elicits specific phenotypes in response to compounds known to interact with membrane lipids, such as papuamide B, amphotericin B and pimaricin. The sensitivity of these novel phenotypes to the genetic modification of the membrane lipid composition was studied by the introduction of the cho1 and lcb1-100 mutations involved respectively in phosphatidylserine or sphingolipid biosynthesis in yeast cells. The results, corroborated by the analysis of equivalent mammalian mutant cell lines, demonstrate that membrane composition, in particular its phosphatidylserine content, influences the function of the transporter. We thus have reconstituted in yeast the essential functions associated to the expression of ABCA1 in mammals and characterized new physiological phenotypes prone to genetic analysis. This article is a part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).

International audience; The ABCA1 transporter orchestrates cellular lipid homeostasis by promoting the release of cholesterol to plasmatic acceptors. The molecular mechanism is, however, unknown. We report here on the biophysical analysis in living HeLa cells of the ABCA1 lipid microenvironment at the plasma membrane. The modifications of membrane attributes induced by ABCA1 were assessed at both the outer and inner leaflet by monitoring either the lifetime of membrane inserted fluorescent lipid analogues by fluorescence lifetime imaging microscopy (FLIM) or, respectively, the membrane translocation of cationic sensors. Analysis of the partitioning of dedicated probes in plasma membrane blebs vesiculated from these cells allowed visualization of ABCA1 partitioning into the liquid disordered-like phase and corroborated the idea that ABCA1 destabilizes the lipid arrangement at the membrane. Specificity was demonstrated by comparison with cells expressing an inactive transporter. The physiological relevance of these modifications was finally demonstrated by the reduced membrane mobility and function of transferrin receptors under the influence of an active ABCA1. Collectively, these data assess that the control of both transversal and lateral lipid distribution at the membrane is the primary function of ABCA1 and positions the effluxes of cholesterol from cell membranes downstream to the redistribution of the sterol into readily extractable membrane pools.