Biology subjects

Lauvau, G.

Publications and source records attributed to Lauvau, G..

12 recordsLinked to original sources

bioRxiv2026

MLL3 adaptor function, not methyltransferase catalytic activity, is essential for breast tumor suppression

MLL3 (Mixed-Lineage Leukemia 3), also known as KMT2C, is one of the most frequently altered epigenetic regulators in breast cancer. MLL3 loss-of-function leads to accelerated tumor onset and growth and increased metastasis. As a large multi-domain protein, MLL3 functions as a histone methyltransferase and a nuclear protein adaptor interacting with other epigenetic proteins. Since breast cancer MLL3 mutations are often truncating mutations that lead to protein degradation, whether the MLL3 tumor suppressor activity depends on its catalytic activity or non-catalytic chromatin adaptor function remains unclear. Here, using CRISPR genetically engineered mouse mammary stem cell organoid-based breast tumor models, we dissected dosage-dependent and domain-specific functions of MLL3 in breast tumor suppression. MLL3 heterozygous loss breast tumor models revealed that MLL3 is haplo-insufficient for breast tumor suppression. Interestingly, homozygous catalytic-dead MLL3-Y4792A mutation did not accelerate tumor onset, growth, or metastasis. By contrast, G367V mutation in the PHD2 domain, which disrupts the BAP1 complex binding without affecting MLL3 protein stability, accelerated tumor onset and growth, phenocopying MLL3 loss. Mechanistically, MLL3 loss impaired chromatin localization of UTX, and genetic depletion of UTX accelerated breast tumor progression in MLL3-wildtype but not MLL3-deficient cells. Integrated RNA-seq, CUT&TAG, and ATAC-seq analyses further showed that transcriptional changes induced by MLL3 loss were more closely associated with promoter-proximal alterations in H3K27Ac, H3K27me3, and chromatin accessibility than with putative MLL3-dependent enhancer regions. Together, these findings reveal that MLL3 suppresses breast tumor initiation through a dosage-sensitive, catalytic-independent adaptor function that regulates promoter-proximal epigenetic states.

cancer biology↗

bioRxiv2024

Intratumoral CXCL12 Gradients Contextualize Tumor Cell Invasion, Migration and Immune Suppression in Breast Cancer

Although the CXCL12/CXCR4 pathway has been prior investigated for its prometastatic and immuno- suppressive roles in the tumor microenvironment, evidence on the spatiotemporal regulation of these hallmarks has been lacking. Here, we demonstrate that CXCL12 forms a gradient specifically around cancer cell intravasation doorways, also known as Tumor Microenvironment of Metastasis (TMEM) doorways, thus facilitating the chemotactic translocation of prometastatic tumor cells expressing CXCR4 toward the perivascular TMEM doorways for subsequent entry into peripheral circulation. Fur- thermore, we demonstrate that the CXCL12-rich micro-environment around TMEM doorways may cre- ate immunosuppressive niches, whereby CD8+ T cells, despite being attracted to these regions, often exhibit reduced effector functions, limiting their efficacy. While the CXCL12/CXCR4 pathway can mini- mally influence the overall composition of immune cell populations, it biases the distribution of CD8+ T cells away from TMEM doorways, justifying its prior-established role as immunosuppressive factor for CD8+ T cells. Our research suggests that the complex interactions between CXCL12 and the various tumor and immune cell types contributes not only to the completion of the initial steps of the metastatic cascade, but also offers an immunological "sanctuary" to prometastatic tumor cells homed around TMEM doorways. Overall, our study enhances our current understanding on the mechanisms, via which CXCL12 orchestrates tumor cell behavior and immune dynamics, potentially guiding future thera- peutic strategies to combat breast cancer metastasis and improve anti-tumor immunity.

cancer biology↗

bioRxiv2023

Prenatal vitamin D deficiency alters immune cell proportions of young adult offspring through alteration of long-term stem cell fates

Vitamin D deficiency is a common deficiency worldwide, particularly among women of reproductive age. During pregnancy, it increases the risk of immune-related diseases in offspring later in life. However, exactly how the body remembers exposure to an adverse environment during development is poorly understood. Herein, we explore the effects of prenatal vitamin D deficiency on immune cell proportions in offspring using vitamin D deficient mice established by dietary manipulation. We found that prenatal vitamin D deficiency alters immune cell proportions in offspring by changing the transcriptional properties of genes downstream of vitamin D receptor signaling in hematopoietic stem and progenitor cells of both the fetus and adults. Our results suggest the role of cellular differentiation properties of the hematopoiesis as the long-term memories of prenatal exposure at the adult stage. Moreover, further investigations of the associations between maternal vitamin D levels and cord blood immune cell profiles from 75 healthy pregnant women and their term babies also confirm that maternal vitamin D levels in the second trimester significantly affect immune cell proportions in the babies. This highlights the importance of providing vitamin D supplementation at specific stages of pregnancy.

developmental biology↗

bioRxiv2022

Loss of the MLL3 tumor suppressor accelerates breast tumor onset via HIF1a-induced CCL2-mediated recruitment of CCR2+ regulatory T cells

While essential gatekeepers of immune homeostasis, Foxp3+ regulatory T (Treg) cells infiltrating tumors acquire distinct phenotypes and become highly immunosuppressive, promoting tumor immune escape and growth. How this occurs and relates to tumor-driver mutations is largely uncharacterized. Herein, we created a mouse mammary stem cell-based tumor model using CRISPR gene editing in which we introduced known human cancer-driver mutations. These included functional loss of the MLL3 histone methyltransferase and p53, and constitutive PI3-kinase activation, recapitulating the genetic makeup of aggressive breast cancers. We show that MLL3 loss fosters tumorigenesis by promoting the rapid establishment of an immunosuppressive microenvironment through induction of HIF1, which increases the secretion of the chemokine CCL2 by tumor cells and the recruitment of higher numbers of Foxp3+ Treg cells via CCR2. Greater infiltration of Treg cells also correlates with MLL3 downregulation and mutations in human breast cancer biopsies. Interestingly, HIF1 enforces the differentiation of tumor-infiltrating Treg cells into highly immunosuppressive ICOShiGITRhi Blimp-1hi effector Treg cells that enable rapid tumor escape. Monoclonal antibody targeting of ICOS or GITR inhibits tumorigenesis in most mice even two months after the cessation of treatment as well as the growth of established tumors, suggesting possible therapeutic opportunities for MLL3-mutant breast cancers.

immunology↗

bioRxiv2022

Chemoenzymatic Measurement of Cell-surface Glycan in Single-cell Multiomics: LacNAc as an Example

Despite the rich information of a cells physiological state encoded in the dynamic changes of cell-surface glycans, methods of capturing glycosylation states at the single-cell level are quite limited. Here we report a chemoenzymatic single-cell N-acetyllactosamine (LacNAc) detection method via tagging the LacNAc with a specific DNA barcode. Compared to the lectin-based glycan detection, the chemoenzymatic labeling does not change the transcriptional status of immune cells and is more compatible with scRNA-seq. Integrated analysis of LacNAc and transcriptome of T cells at a single-cell level reveals that the quantity of cell-surface LacNAc is significantly upregulated in activated CD8+ T cells but maintained at the basal level in quiescent CD8+ T cells (i.e., naive and central memory T cells). Further analysis confirms that the LacNAc level is positively correlated to the glycolytic activity of CD8+ T cells at all statues. Taken together, our study demonstrates the feasibility of chemoenzymatic detection of cell-surface glycan in single-cell RNA sequencing-based multiomics with information of TCR sequence and cell-surface epitopes (i.e., scTCR and CITE-seq) and offers a new way to characterize the biological role of glycan in diversified physiological states.

immunology↗

bioRxiv2022

CD169+ macrophages orchestrate plasmacytoid dendritic cell arrest and retention for optimal priming in the bone marrow of malaria-infected mice

Plasmacytoid dendritic cells (pDC) are the most potent producer of type I interferon (IFN), but how pDC are primed in vivo is poorly defined. Using a mouse model of severe malaria, we have previously established that upon priming by CD169+ macrophages (MP), pDC initiate type I IFN-I secretion in the bone marrow (BM) of infected mice via cell-intrinsic TLR7 sensing and cell-extrinsic STING sensing. Herein we show that CD169+ MP and TLR7-sensing are both required for pDC arrest during priming, suggesting CD169+ MP are the source of TLR7 ligands. We establish that TLR7 sensing in pDC and chemotaxis are both required for pDC arrest and functional clustering with CD169+ MP in the BM. Lastly, we demonstrate that STING-sensing in CD169+ MP control pDC initiation of type I IFN production while also regulating pDC clustering and egress from the BM. Collectively, these results link pDC acquisition of type I IFN secreting capacity with changes in their motility, homing and interactions with CD169+ MP during infection. Thus, targeting this cellular interaction may help modulate type I IFN to improve outcomes of microbial infections and autoimmune diseases.

immunology↗

bioRxiv2021

T cell receptor and IL-2 signaling strength control memory CD8+ T cell functional fitness via chromatin remodeling

Cognate antigen signal controls CD8+ T cell priming, expansion size and effector versus memory cell fates, but it is not known if and how it modulates the functional features of memory CD8+ T cells. Here we show that the strength of T cell receptor (TCR) signaling determines the requirement for interleukin-2 (IL-2) signals to form a pool of memory CD8+ T cells that competitively re-expand upon secondary antigen encounter. Combining strong TCR and intact IL-2 signaling synergistically induces genome-wide chromatin accessibility in regions targeting a wide breadth of biological processes, consistent with their greater functional fitness. Chromatin accessibility in promoters of genes encoding for stem cell, cell cycle and calcium-related proteins correlated with faster intracellular calcium accumulation, initiation of cell cycle and more robust expansion. High-dimensional flow-cytometry analysis also highlights higher subset diversity and phenotypes. These results formally establish that epitope selection in vaccine design strongly impacts memory CD8+ T cell epigenetic programming and functions. One Sentence SummaryThe strength of antigenic and interleukin 2 signals received by CD8+ T cells during vaccination epigenetically programs their ability to form functional memory.

immunology↗

bioRxiv2021

Cytolytic memory CD4+ T cell clonotypes are expanded during Plasmodium falciparum infection

Plasmodium falciparum (Pf) malaria causes high rates of morbidity and mortality and lacks a sufficiently effective vaccine. Clinical immunity develops in residents of malaria endemic regions which confers reduced clinical symptoms during infection and protection against severe disease. We hypothesized that understanding the immune mechanisms of clinical immunity could inform vaccine design to improve efficacy. We compared the peripheral blood cellular and humoral immune responses during a mild episode of Pf malaria infection. Participants were classified as either clinically susceptible or clinically protected, based on the number of recurrent clinical infections over an 18-month longitudinal study in a malaria endemic region in Malawi. Susceptible participants had three or more recurrent clinical episodes while clinically immune individuals had one or none. Protected participants exhibited higher plasma immunoglobulin G (IgG) breadth and titers against Pf antigens, and greater antibody (Ab)-dependent Pf opsonization compared to susceptible participants. Using high dimensional mass cytometry (CyTOF), spectral flow cytometry and single-cell transcriptomic analyses, we identified expanded memory CD4+ T cell clones sharing identical T cell receptor clonotypes in the blood of protected participants during malaria infection. These cells express a strong cytolytic T helper 1 effector program with transcripts encoding granzymes (A, B, H, M), granulysin, NKG7 and the Zeb2 master transcriptional regulator of terminally differentiated effector T cells. Memory CD4+ T cells expressing Zeb2+ were CD39hiTIGIThi and expressed multiple chemotactic and checkpoint inhibitory receptors, although the cellular levels of several of these receptors were reduced in protected compared to susceptible individuals. We propose that clonally expanded Zeb2+ cytolytic memory CD4+ Th1 cells could represent essential contributors to clinical immunity against Pf malaria. One Sentence SummaryA population of cytolytic memory CD4+ T cells is clonally expanded in patients with Plasmodium falciparum malaria and has reduced chemotactic and inhibitory receptor expression in patients with naturally acquired clinical malaria immunity.

immunology↗

bioRxiv2021

Memory CD8+ T cells mediate early pathogen-specific protection through localized delivery of chemokines and IFNγ to clusters of inflammatory monocytes

While cognate antigen drives clonal expansion of memory CD8+ T cells to achieve sterilizing immunity in immunized hosts, not much is known on how cognate antigen contributes to early mechanisms of protection before clonal expansion occurs. Herein, using distinct models of immunization, we establish that cognate antigen recognition by CD8+ TM cells on dendritic cells initiates their rapid and coordinated production of a burst of CCL3, CCL4 and XCL1 chemokines under the transcriptional control of IRF4. Using intravital microscopy imaging and in vivo monoclonal antibody labelling, we reveal that memory CD8+ T cells undergo antigen-mediated arrest in splenic red pulp clusters of CCR2+ monocytes where they locally deliver both IFN{gamma}- and chemokine-potentiating microbicidal activities to achieve early protection. Thus, rapid and effective memory CD8+ T cell responses require a complex series of spatially and temporally coordinated stepwise molecular and cellular events that quickly restrict microbial pathogen growth and optimize the local delivery of effector molecules before clonal expansion occurs.

immunology↗

bioRxiv2020

Blockade of LAG-3 in PD-L1-deficient mice enhances clearance of blood stage malaria independent of humoral responses

T cells expressing high levels of inhibitory receptors such as PD-1 and LAG-3 are a hallmark of chronic infections and cancer. Checkpoint blockade therapies targeting these receptors have been largely validated as promising strategies to restore exhausted T cell functions and clearance of chronic infections and tumors. The inability to develop long-term natural immunity in malaria-infected patients has been proposed to be at least partially accounted for by sustained expression of high levels of inhibitory receptors on T and B lymphocytes. While blockade or lack of PD-1/PD-L1 and/or LAG-3 was reported to promote better clearance of Plasmodium parasites in mice, how exactly these pathways contributes to protection is not known. Herein, using a mouse model of non-lethal P. yoelii (Py) infection, we reveal that the kinetics of blood parasitemia is indistinguishable between PD-1-/-, PD-L1-/- and WT mice. Yet, monoclonal antibody (mAb) blockade of LAG-3 in PD-L1-/- mice promoted accelerated control of blood parasite growth and clearance. We also report that i) the majority of LAG-3+ cells are T cells, ii) selective depletion of CD8+ T cells did not prevent anti-LAG-3-mediated protection, and iii) production of effector cytokines by CD4+ T cells is increased in anti-LAG-3-treated versus control mice. In addition, parasite-specific Ab serum titers and their ability to transfer protection from both groups of mice was comparable and depletion of CD4+ T cells prevented protection. Thus, taken together, these results are consistent with a model in which disruption of PD-L1 and LAG-3 on parasite-specific CD4+ T cells unleashes their ability to effectively clear blood parasites, independently from humoral responses. Author SummaryMalaria, caused by Plasmodium parasites, is a global burden for which an efficacious vaccine is urgently needed. The development of long-term immunity against malaria is unclear, but we know that both T and B (that produce antibodies, Ab) lymphocytes, that are subsets of white blood cells, are required. Studies in mouse models of malaria have suggested that sets of inhibitory receptors, namely LAG-3 and PD-1, expressed on cytotoxic and helper T lymphocytes hamper the development of effective immunity against malaria. Therapeutic blockade of these receptors was reported to enhance blood parasite clearance through the development of more protective parasite-specific helper T lymphocytes and Abs. Herein, we reveal that, while mice genetically deficient for the PD-1 pathway fail to clear blood parasites better than WT counterparts, anti-LAG-3 treatment does. Importantly, we found comparable parasite-specific Ab responses between all mouse groups, and Ab transfers conferred similar protection to newly infected mice. We also show that LAG-3 is mostly expressed on T lymphocytes, and that cytotoxic T lymphocytes are not involved in anti-LAG-3 accelerated clearance of parasites. Our results suggest that LAG-3 blockade acts on helper T lymphocytes to unleash their effector responses and enhance the control of blood-stage malaria, independently from parasite-specific Abs.

immunology↗

bioRxiv2018

Single-step Enzymatic Glycoengineering for the Construction of Antibody-cell Conjugates

Employing live cells as therapeutics is a direction of future drug discovery. An easy and robust method to modify the surfaces of cells directly to incorporate novel functionalities is highly desirable. However, many current methods for cell-surface engineering interfere with cells endogenous properties. Here we report an enzymatic approach that enables the transfer of biomacromolecules, such as a full length IgG antibody, to the glycocalyx on the surfaces of live cells when the antibody is conjugated to the enzymes natural donor substrate GDP-fucose. This method is fast and biocompatible with little interference to cells endogenous functions. We applied this method to construct two antibody-cell conjugates (ACCs) using different immune cells, and the modified cells exhibited specific tumor targeting and resistance to inhibitory signals produced by tumor cells, respectively. Remarkably, Herceptin-NK-92MI conjugates exhibits enhanced activities to induce the lysis of HER2+ cancer cells both ex vivo and in a murine tumor model, indicating its potential for further development as a clinical candidate.

bioengineering↗

bioRxiv2017

Listeria monocytogenes virulence factors are secreted in biologically active Extracellular Vesicles

Outer membrane vesicles produced by Gram-negative bacteria have been studied for half a century but the possibility that Gram-positive bacteria secreted extracellular vesicles (EVs) was not pursued due to the assumption that the thick peptidoglycan cell wall would prevent their release to the environment. However, following discovery in fungi, which also have cell walls, EVs have now been described for a variety of Gram-positive bacteria. EVs purified from Gram-positive bacteriaare implicated in virulence, toxin release and transference to host cells, eliciting immune responses, and spread of antibiotic resistance. Listeria monocytogenes is a Gram-positive bacterium that is the etiological agent of listeriosis. Here we report that L. monocytogenes produces EVs with diameter ranging from 20-200 nm, containing the pore-forming toxin listeriolysin O(LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC). Using simultaneous metabolite, protein, and lipid extraction (MPLEx) multi-omics we characterized protein, lipid and metabolite composition of bacterial cells and secreted EVs and found that EVs carry the majority of listerial virulence proteins. Cell-free EV preparations were toxic to the murine macrophage cell line J774.16, in a LLO-dependent manner, evidencing EV biological activity. The deletion of plcA increased EV toxicity, suggesting PI-PLC can restrain LLO activity. Using immunogold electron microscopy we detect LLO localization at several organelles within infected human epithelial cells and with high-resolution fluorescence imaging we show that dynamic lipid structures are released from L. monocytogenes that colocalize with LLO during infection. Our findings demonstrate that L. monocytogenes utilize EVs for toxin release and implicate these structures in mammalian cytotoxicity.

microbiology↗