Biology subjects

Bousso, P.

Publications and source records attributed to Bousso, P..

11 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

The Pentose Phosphate Pathway Regulates Myelo-Lymphoid Lineage Specification

Following infection, hematopoietic stem and progenitor cells (HSPCs) support immunity by increasing the rate of innate immune cell production but the metabolic cues that guide this process are unknown. To address this question, we combined in situ RNA barcoding and metabolomics approaches to perform metabolic state-fate mapping in vivo. This approach revealed a subset of myeloid-biased HSPCs that express a distinct set of metabolic enzymes and transporters as well as the surface marker CD62L. Metabolically, CD62Lhigh HSPCs have differential activity of the pentose phosphate pathway (PPP), OXPHOS and translation, as well as differential levels of S-adenosylmethionine (SAM) cycle metabolites associated with epigenetic modifications. Inhibition of the PPP skews HSPC lineage fate decisions by disrupting myeloid associated enhancers, while simultaneously increasing enhancer activity for the master B-lymphoid regulator Ikaros. In vivo, overexpression of glucose-6-phosphate dehydrogenase, a rate limiting enzyme of the PPP, skewed HSPC output from B-lymphocytes. In summary, our data shows that HSPCs undergo significant metabolic changes to facilitate the bioenergetic and epigenetic demands of myeloid versus lymphoid lineage specification. We highlight a key role for the pentose phosphate pathway which modulates myeloid rather than lymphoid commitment by shaping the HSPC enhancer landscape, providing proof of principle that HSPC metabolism can be targeted to modulate immune system dynamics.

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

Met-Vision stratifies single cell metabolism revealing coexisting energetic states in tissue macrophages redistributed by inflammation

The function of tissue-associated macrophages is tightly linked to their energy metabolism. Yet, the diversity of macrophage metabolic profiles coexisting in tissues at homeostasis or during immune challenges is incompletely understood. Here, we introduce Met-Vision, an imaging-based pipeline for single-cell functional profiling and classification of energy metabolism. Across multiple tissue contexts, we identified that macrophages do not adopt a uniform metabolic profile but typically co-exist in four discrete metabolic states with distinct dependence on OXPHOS and metabolic plasticity. Inflammation reconfigured the distribution of macrophage metabolic profiles that remained heterogeneous. Notably, inflammation-derived nitric oxide finely tuned the distribution of macrophage energetic states. These findings challenge the view of homogenous metabolic activation and reveal a layer of metabolic diversity in tissue at steady state and during inflammation. The ability to stratify macrophage energy metabolic profiles with Met-Vision should help guide the development of metabolism-targeted therapies for inflammatory diseases, cancer, and metabolic disorders. Significance statementThis study introduces Met-Vision and reveal that tissue macrophages occupy four distinct energetic states, challenging the notion of uniform metabolic activation and opening avenues for guiding metabolism-targeted therapies.

immunology↗

bioRxiv2025

Loss of the tumor suppressor PTEN activates cell-intrinsic interferon signaling to drive immune resistance

Loss of the tumor suppressor PTEN is strongly associated with a lack of response to immune checkpoint blockade therapies in cancer patients, but the underlying mechanisms are not fully understood. We have developed a transformation model where knocking out PTEN in human mammary epithelial cells drives dependence on the p110{beta} subunit of PI3K and, notably, elicits a robust induction of endogenous retroviral elements (ERVs) and activation of the interferon signaling. This constitutive cell-intrinsic interferon response, hallmarked by hyperactivated STAT1, is also observed in human tumors with a PTEN-low status. We further find that PTEN deficiency renders cancer cells resistant to the cytotoxic effects of immune cells and interferon-{gamma}. Notably, PTEN loss also results in a dependency on an activated DNA damage response pathway, leading to an exquisite vulnerability to CDK12 inhibition. Our study suggests an interferon adaptation model in which tumors driven by PTEN deficiency inherently activate the interferon response, enabling them to adapt to interferon cytotoxicity and gain resistance to immunotherapies.

cancer biology↗

bioRxiv2023

Metabolically Primed Multipotent Hematopoietic Progenitors Fuel Innate Immunity

Following infection, hematopoietic stem and progenitor cells (HSPCs) support immunity by increasing the rate of innate immune cell production but the metabolic cues that guide this process are unknown. To address this question, we developed MetaFate, a method to trace the metabolic expression state and developmental fate of single cells in vivo. Using MetaFate we identified a gene expression program of metabolic enzymes and transporters that confers differences in myeloid differentiation potential in a subset of HSPCs that express CD62L. Using single-cell metabolic profiling, we confirmed that CD62Lhigh myeloid-biased HSPCs have an increased dependency on oxidative phosphorylation and glucose metabolism. Importantly, metabolism actively regulates immune-cell production, with overexpression of the glucose-6-phosphate dehydrogenase enzyme of the pentose phosphate pathway skewing MPP output from B-lymphocytes towards the myeloid lineages, and expansion of CD62Lhigh HSPCs occurring to support emergency myelopoiesis. Collectively, our data reveal the metabolic cues that instruct innate immune cell development, highlighting a key role for the pentose phosphate pathway. More broadly, our results show that HSPC metabolism can be manipulated to alter the cellular composition of the immune system.

immunology↗

bioRxiv2022

Leishmania amazonensis controls macrophage-regulated cell death to establish chronic infection in vitro and in vivo

Pathogenic protists of the genus Leishmania have evolved various strategies to exploit macrophages as host cells and subvert their immuno-metabolic functions to favour intracellular parasite survival. Surprisingly little is known on how Leishmania affects regulated cell death (RCD) pathways of its host cell, even though increased survival of in vitro infected macrophages has been reported, and chronic macrophage infection in vivo causes the devastating immunopathologies of leishmaniasis. To overcome this limitation and gain first systems-level insight into the interaction between intracellular Leishmania and the host cell RCD pathways, including apoptosis, pyroptosis and necroptosis, we applied transcriptomic analyses on L. amazonensis-infected, primary macrophages (termed LIMs) and used YO-PRO-1 to monitor cell death by fluorescent microscopy. RNAseq analyses at day 3 post-infection (PI) revealed dichotomic dysregulation of more than 60% of RCD-related genes in LIMs, characterized by up-regulation of anti-RCD and down-regulation of pro-RCD markers, including key regulators common to the three forms of cell death such as casp8, fadd, tradd, tnfaip3, tax1bp1, birc3, and itch. This profile correlated with expression changes of transcription factors known to regulate RCD, including AP1 and NF-{kappa}B family members, ppar{gamma} and cebp{beta}. Consequently, LIMs showed remarkable longevity in culture for at least 50 days, despite a constant increase of parasite burden to about 100 parasites per cell, while non-infected cells were cleared from the culture in just a few days. Longitudinal expression analysis of LIMs at days 0, 3, 15, and 30 PI by RT-qPCR confirmed stable maintenance of this high longevity profile with the dichotomic decrease and increase of RCD-activators and -inhibitors, respectively. LIMs further showed significant resistance to RCD-inducing signals compared to non-infected cells, including CSF-1 deprivation (intrinsic apoptosis), actinomycin D treatment (extrinsic apoptosis), LPS/ATP stimulation (pyroptosis). Significantly, we extended the anti-RCD expression pattern and RCD resistance phenotype to L. amazonensis-infected macrophages recovered from lesions, thus validating our long-term in vitro infection system as an easily accessible model to study chronic macrophage infection. In conclusion, our analyses firmly document the pan-anti RCD effect of L. amazonensis on its macrophage host cell in vitro and in vivo and shed important new light on mechanisms underlying Leishmania chronic infection.

microbiology↗

bioRxiv2022

Immunometabolic profiling of in vitro and ex vivo Leishmania-infected macrophages (LIMs) reveals unique polarization and bioenergetic signatures

Macrophages are the major host cells of the protozoan parasite Leishmania in mammalian infection. These key innate immune cells display remarkable phenotypic plasticity ranging from pro-inflammatory M1 to anti-inflammatory M2 macrophages that can control infection and tissue homeostasis, respectively. It has been recognized that Leishmania exploits macrophage phenotypic plasticity to establish chronic infection. However, the current notion that these parasites simply trigger an M2-like phenotype seems over-simplified considering the immunopathology observed during leishmaniasis - in particular in response to Leishmania amazonensis - which is often characterized by a mixed Th1/Th2 immune response. Here we combined a series of systems-level analyses to shed new light on the phenotype of Leishmania-infected macrophages (LIMs) during short- and long-term infection, in vitro and in vivo. Immuno-metabolic profiling by RNA-seq, RT-qPCR, cytokine immunoassays, and real-time bioenergetic flux analysis of L. amazonensis-infected bone marrow-derived macrophages (BMDMs) revealed a highly complex and unique phenotypic and bioenergetic signature. In vitro LIMs were characterized by co-expression of both M1 and M2 markers at RNA and protein levels and increased expression of glycolytic genes that matched a progressive metabolic switch from a M2-like respiratory to a M1-like glycolytic energy production observed for both long-term in vitro and in vivo infected macrophages. Unlike in M1 macrophages, glycolytic gene expression did not correlate with increased expression of its key regulatory HIF-1. In contrast, siRNA knock down experiments in primary BMDMs uncovered an essential role of the m6A reader protein IGF2BP2 in stabilizing m6A modified transcripts of the glycolytic pathway, contributing to HIF-1-independent induction of glycolysis. In conclusion, L. amazonensis establishes a complex and unique phenotypic shift in infected macrophages in vitro and in vivo that combines M1-like and M2-like immuno-metabolomic characteristics and implicates differential mRNA stability in induction of aerobic glycolysis. Our data thus uncover epi-transcriptomic regulation as a novel target for Leishmania immune subversion to establish a host cell phenotype beneficial for intracellular parasite development and chronic infection.

immunology↗

bioRxiv2021

A Multiscale Immuno-Oncology on-Chip System (MIOCS) establishes that collective T cell behaviors govern tumor regression

T cell-based tumor immunotherapies such as CAR T cells or immune checkpoint inhibitors harness the cytotoxic potential of T cells to promote tumor regression. However, patient response to immunotherapy remains heterogeneous, highlighting the need to better understand the rules governing a successful T cell attack. Here, we develop a microfluidic-based method to track the outcome of T cell activity on many individual cancer spheroids simultaneously, with a high spatiotemporal resolution. By combining these parallel measurements of T cell behaviors and tumor fate with probabilistic modeling, we establish that the first recruited T cells initiate a positive feedback loop leading to an accelerated effector accumulation on the spheroid. We also provide evidence that cooperation between T cells on the spheroid during the killing phase facilitates tumor destruction. We propose that tumor destruction does not simply reflect the sum of individual T cell activities but relies instead on collective behaviors promoting both T cell accumulation and function. The possibility to track many replicates of immune-tumor interactions with such a level of detail should help delineate the mechanisms and efficacy of various immunotherapeutic strategies.

biophysics↗

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↗

bioRxiv2018

Macropinocytosis overcomes directional bias due to hydraulic resistance to enhance space exploration by dendritic cells

The migration of immune cells is guided by specific chemical signals, such as chemokine gradients. Their trajectories can also be diverted by physical cues and obstacles imposed by the cellular environment, such as topography, rigidity, adhesion, or hydraulic resistance. On the example of hydraulic resistance, it was shown that neutrophil preferentially follow paths of least resistance, a phenomenon referred to as barotaxis. We here combined quantitative imaging and physical modeling to show that barotaxis results from a force imbalance at the scale of the cell, which is amplified by the acto-myosin intrinsic polarization capacity. Strikingly, we found that macropinocytosis specifically confers to immature dendritic cells a unique capacity to overcome this physical bias by facilitating external fluid transport across the cell, thereby enhancing their space exploration capacity in vivo and promoting their tissue-patrolling function. Conversely, mature dendritic cells, which down-regulate macropinocytosis, were found to be sensitive to hydraulic resistance. Theoretical modeling suggested that barotaxis, which helps them avoid dead-ends, may accelerate their migration to lymph nodes, where they initiate adaptive immune responses. We conclude that the physical properties of the microenvironment of moving cells can introduce biases in their migratory behaviors but that specific active mechanisms such as macropinocytosis have emerged to diminish the influence of these biases, allowing motile cells to reach their final destination and efficiently fulfill their functions.

cell biology↗