Biology subjects

Einsele, H.

Publications and source records attributed to Einsele, H..

15 recordsLinked to original sources

bioRxiv2026

Tumor-tropic E. coli engineered as living T and NK cell engagers

Despite advances in immunotherapy, most solid tumors remain resistant to treatment. Immune cell engagers redirect cytotoxic lymphocytes against cancer, but limited tumor access, immunosuppressive microenvironments and systemic immune activation limit efficacy. Here we develop live immune modulating engagers (LIME), a modular platform where non-pathogenic, tumor-tropic Escherichia coli display tandem single-chain variable fragments targeting a tumor-associated antigen and an activating receptor on T or natural killer cells. LIME bridged effector and tumor cells, induced transcriptional programs of T cell activation, metabolism and proliferation, and enhanced cytotoxicity across cancer cell lines and patient-derived organoids. In mouse models, LIME safely accumulated in tumors, outperformed tarlatamab in small cell lung cancer, and induced durable immunity in lymphoma. RAS inhibition and PD-L1 blockade enhanced LIME activity in pancreatic cancer and induced humoral responses. Multi-lineage immune modulation remained tumor-confined, without organ toxicity. These findings establish LIME as a versatile living therapeutic platform for programmable, tumor-restricted immune orchestration.

bioengineering↗

bioRxiv2026

M-CSF stimulated alveolar macrophages safeguard from invasive aspergillosis

Invasive pulmonary aspergillosis (IPA) is a life-threatening complication in immunocompromised individuals, including recipients of allogeneic hematopoietic cell transplantation (allo-HCT). While systemic neutropenia is traditionally considered the primary risk factor for IPA, we demonstrate that tissue-resident alveolar macrophages (AMs), rather than recruited neutrophils, dictate survival during the critical early window after transplantation. Utilizing an ultra-low dose Aspergillus fumigatus infection model that mimics physiological exposure, we identify alveolar macrophages (AMs) as key players in pulmonary antifungal defense. In immunocompromised mice, AMs conferred protection against lethal invasive aspergillosis by day 6, but not day 4 post-allo-HCT. To enhance AM function at the earlier time point, we tested cytokine-based interventions and show that M-CSF, but not IL-34, which both bind to the CSF-1 receptor, promotes migratory activity, phagolysosomal function and fungal killing in both mouse and human primary tissue-resident AMs. In allo-HCT recipient mice, M-CSF treatment preserved lung tissue integrity, suppressed pro-inflammatory cytokines, and protected mice from lethal invasive aspergillosis. The M-CSF-driven protective effect was abrogated upon AM depletion. Our findings demonstrate a critical role of tissue-resident AMs in pulmonary antifungal immunity and suggest that therapeutic modulation of AM activity via M-CSF may offer a promising strategy to combat severe fungal infections in immunocompromised patients. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/736478v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@49ba37org.highwire.dtl.DTLVardef@607c4forg.highwire.dtl.DTLVardef@814894org.highwire.dtl.DTLVardef@1c413c9_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2026

Profound CD4+ T-Cell Reprogramming by Melphalan-Driven Oxidative Stress in High-Risk Multiple Myeloma

T cell-based immunotherapies have become central to the treatment of multiple myeloma (MM), yet their efficacy depends on the functionality of endogenous T cells. How cumulative treatment exposure, particularly high-dose melphalan, together with disease-intrinsic high-risk features shapes T-cell composition and immune competence remains incompletely understood. Here, we analyzed T cell composition and function in bone marrow (BM) and peripheral blood (PB) samples from MM patients across different stages of their treatment journey using flow cytometry (BM, n=162; PB, n=1,733), single-cell RNA sequencing (n=19), and cytotoxicity assays (n=20). We reveal reduced overall T cell frequencies and CD4+/CD8+ T cell ratio, associated with lines of therapy and driven in part by depletion of naive CD4+ T cells in gene-expression defined high risk (HR) disease. Among therapeutic agents, melphalan exerted the strongest effects on T cell populations and induced pronounced redox stress in both T cells and myeloma cell lines. This oxidative stress signature was enriched in HR patients and was reversible with N-acetyl-L-cysteine treatment. Together, these findings identify immune dysregulation as a defining feature of HR MM that extends beyond tumor-intrinsic genomic alterations and is further shaped by treatment-induced remodeling of the BM microenvironment. Given the association between higher CD4+ T cell numbers and improved CAR-T cell outcomes, our data highlight the translational importance of treatment sequencing, particularly in HR MM. One Sentence SummaryMelphalan-induced redox stress depletes CD4+ naive T cells, particularly in patients with high-risk multiple myeloma.

Cancer Biology↗

bioRxiv2026

Integrative phenotypic and transcriptomic validation of an alveolar-like macrophage model reveals early host-pathogen dynamics during Aspergillus fumigatus infection

Primary alveolar macrophages (pAMs) are essential for the rapid clearance of conidia and maintenance of pulmonary homeostasis. However, Aspergillus fumigatus remains the leading cause of invasive pulmonary aspergillosis in immunocompromised patients, and the mechanisms governing fungal clearance versus invasion remain poorly understood. Although, pAMs can be isolated from human donors, their broader application in in vitro infection studies is limited by their low availability and technical challenges with their maintenance in culture. In this study, we successfully adapted a previously established monocyte-derived alveolar-like macrophage (ALM) model to investigate early host-pathogen interactions upon A. fumigatus challenge. Given the requirement of GM-CSF for maintaining alveolar macrophage identity and function, we included GM-CSF differentiated macrophages (GM-M), as a widely employed reference model. Primary alveolar macrophages (pAM), isolated from human lung biopsies were utilized to validate the physiological relevance of the ALM model. Combined phenotypic, transcriptomic and functional analyses demonstrated that ALMs closely resemble pAMs under both steady-state and infection conditions across multiple time points and fungal burdens. Notably, fungal dual RNA-sequencing revealed a significant upregulation of fungal virulence-associated factors during interaction with ALM, which was not observed in our GM-M co-cultures. Collectively, these findings support the use of ALMs as a robust, experimentally accessible and physiologically relevant in vitro model for investigating early A. fumigatus infection, providing new insights into host-pathogen dynamics at the alveolar interface.

microbiology↗

bioRxiv2025

Secondary lymphoid organ endothelial cells prime alloreactive CD4+ T cells to trigger acute graft-versus-host disease

Donor CD4 T cell priming is a pivotal determinant of acute graft-versus-host disease (aGvHD) after allogeneic hematopoietic cell transplantation (allo-HCT). While professional hematopoietic antigen-presenting cells (APCs) have long been implicated in the pathogenesis of aGvHD, the contribution of non-hematopoietic APCs has remained unclear. Here, we show that naive alloreactive CD4 T cells initially localize and activate specifically within secondary lymphoid organs (SLOs) before infiltrating target tissues. Using genetic models to selectively ablate MHC class II on endothelial cells (ECs) or hematopoietic cells, we demonstrate that blood endothelial cells (BECs) in SLOs function as APCs, efficiently processing and presenting antigen to prime donor CD4 T cells. Deletion of MHC class II (MHCII) specifically in ECs substantially attenuates T cell activation and protects mice from lethal aGvHD, whereas selective deletion of MHCII in lymphatic ECs has no effect. Likewise, selective deletion of MHCII in hematopoietic cells also protects mice against aGvHD, suggesting that both cell types contribute to pathogenic allogeneic T cells activation after allo-HCT. Mechanistically, IL-12/IFN{gamma} signaling upregulates MHC class II expression on BECs. These findings identify BECs in SLOs as initiators of alloreactive CD4 T cell responses and highlight a potential target for preventing aGvHD. HighlightsO_LIBlood endothelial cells in secondary lymphoid organs prime naive CD4 T cells to trigger acute GvHD. C_LIO_LIT cell activation occurs exclusively in secondary lymphoid organs before tissue infiltration. C_LIO_LIExpression of MHC class II only on endothelial cells is sufficient to drive lethal GvHD, independent of other antigen presenting cells. C_LIO_LIRegulation of MHC class II expression in blood endothelial cells by IL-12/IFN{gamma} offers the potential for new therapeutic targets and corroborates findings for existing therapeutics. C_LI

immunology↗

bioRxiv2025

Single-molecule localization microscopy reveals the molecular organization of endogenous membrane receptors

Super-resolution microscopy in combination with genetic labeling methods allows imaging of single proteins in cells. However, visualizing endogenous proteins on primary cells remains challenging due to the use of sterically demanding antibodies for labeling. Here, we demonstrate how immunolabeling conditions and antibody crosslinking influence the quantification and identification of membrane receptor stoichiometry on cells using single-molecule localization microscopy. We developed an optimized immunolabeling and analysis protocol and demonstrate the performance of the approach by resolving the molecular organization of endogenous CD45, CD69, and CD38 on Jurkat T cells. To demonstrate the usefulness of the method for immunotherapy applications we investigated the interaction of primary multiple myeloma cells with the therapeutic monoclonal antibodies (mAbs) daratumumab and isatuximab, and a polyclonal anti-CD38 antibody. Our approach might lay the foundation for improved personalized diagnostics and treatment with therapeutic antibodies. One-Sentence SummarySingle-molecule localization microcopy quantifies the expression and resolves the stoichiometry of endogenous membrane receptors

biophysics↗

bioRxiv2025

Unveiling immune interference: How the dendritic cell response to co-infection with Aspergillus fumigatus is modulated by human cytomegalovirus and its virokine CMVIL-10

Human cytomegalovirus (HCMV) is a master of immune evasion and a potent modulator of the human immune system. The best-characterized mechanism employed by HCMV to suppress host immunity is the production of a viral interleukin-10 homolog (CMVIL-10). While CMVIL-10 is known to suppress immune responses and promote viral persistence, its capacity to promote increased susceptibility to co-infecting pathogens like Aspergillus fumigatus remains unknown. Therefore, we studied the impact of wild-type (WT) HCMV (strain TB40 BAC4), a CMVIL-10-deficient HCMV mutant ({Delta}UL111A), and recombinant CMVIL-10 on the immune activity of monocyte-derived dendritic cells (moDCs) during co-infection with A. fumigatus. Using a combination of transcriptomic and phenotypic readouts, our data revealed a strong and time-dependent immuno-paralytic effect of HCMV by suppressing pathogen recognition pathways, cytokine production, DC maturation, and expression of genes that are essential for host defense and tissue repair. Although infection with {Delta}UL111A lacking CMVIL-10 led to stronger expression of type I interferons, IFN-{gamma}-inducible chemokines, and proinflammatory cytokines than WT infection, interference with antifungal immune defense and fungal clearance during co-infection was largely similar between both strains. The limited effect of CMVIL-10 on antifungal immune defense persisted even after prolonged pre-exposure of DCs to the recombinant virokine. In summary, although CMVIL-10 contributes to shaping an anti-inflammatory environment, HCMVs suppression of antifungal immunity appears to be multifactorial, with CMVIL-10 alone playing a rather subtle role in altering DC responses to A. fumigatus during viral-fungal co-infection. ImportanceHuman cytomegalovirus (HCMV) is a highly prevalent herpesvirus that establishes lifelong latency and frequently reactivates in immunocompromised individuals, including hematopoietic stem cell transplant recipients. Reactivation not only causes direct disease but also increases the risk of secondary infections, such as invasive pulmonary aspergillosis caused by Aspergillus fumigatus. Specifically, studies estimated that about 6-25% of critically ill HCMV-positive patients develop HCMV-associated pulmonary aspergillosis. However, the mechanisms by which HCMV creates a permissive environment for fungal superinfection remain poorly understood. HCMV encodes a viral homolog of interleukin-10 (CMVIL-10), which mimics host IL-10 and elicits potent immunomodulatory activity. Here, we show that CMVIL-10 dampens specific anti-viral responses, DC activation, and cytokine signaling. However, HCMV-mediated impairment of fungal control in co-infection settings occurred largely independent of CMVIL-10 expression. These findings suggest that HCMV undermines antifungal defenses through multifactorial mechanisms beyond CMVIL-10, highlighting the need for targeted strategies to restore immune function in high-risk patients.

immunology↗

bioRxiv2025

A novel CAR T cell blend targeting PDPN and GD2 to overcome glioblastoma heterogeneity

BackgroundWhile chimeric antigen receptor (CAR) T cells have achieved encouraging remission rates in hematological malignancies, they demonstrated limited success in treating Glioblastoma (GBM), particularly due to high intra- and intertumoral heterogeneity. In this study, we identified a relevant and preserved target antigen, Podoplanin (PDPN), and evaluated the potential of a PDPN- and GD2-CAR T cell blend to overcome GBM heterogeneity. MethodsTarget antigen screening included clinical samples, healthy tissues and cell lines, as well as publicly available RNA sequencing datasets. The anti-tumor function of CAR T cells were examined in co-culture experiments with GBM cell lines and patient-derived organoids (PDOs), and in vivo after locoregional delivery in orthotopic xenograft models. ResultsThe generated CAR T cells demonstrated strong anti-tumor activity against several cell lines and PDOs from multiple patients. PDPN and GD2 expression was detectable in all PDOs at varying densities and regardless of the antigenic profile, the CAR T cell blend induced significantly higher levels of apoptosis in organoids than single antigen targeting counterparts. In vivo, we observed efficient tumor regression after locoregional administration of monospecific CAR T cells. While heterogeneous orthotopic tumors eventually relapsed in these groups, blended therapy resulted in a significantly increased overall survival and even achieved cure in the majority of mice. ConclusionThis novel PDPN-/GD2-CAR T cell blend demonstrated strong efficacy in advanced preclinical models of glioblastoma. The results suggest that this approach can overcome GBM heterogeneity in clinical application and address previous limitations of single antigen CAR T cell therapies. KEYPOINTSO_LIPDPN is a relevant and consistent CAR T cell target antigen in primary and recurrent GBM C_LIO_LIPDPN- and GD2-CAR T cells display synergistic anti-tumor activity in patient-derived organoids C_LIO_LILocal delivery of our CAR T cell blend confers long-term survival and cure in GBM-bearing mice C_LI IMPORTANCE OF THE STUDYThe highly variable landscape of tumor antigens in GBM represents a serious obstacle to single antigen CAR T cell therapies. In this study, we identified Podoplanin (PDPN) and GD2 as the most preserved target antigens in a screening campaign, with an even increased density in recurrent GBM compared to primary GBM. We combined PDPN- and GD2-CAR T cells in a blended treatment approach to counter antigen heterogeneity, and achieved a substantial gain in efficacy against patient-derived organoids (PDOs). In an orthotopic xenograft model, locoregional administration of the PDPN- and GD2-CAR T cell blend conferred long-term complete remission and increased survival compared to single antigen targeting. This study provides a hierarchy of CAR target antigens for treating GBM and illustrates the therapeutic potential of combinatorial antigen targeting using a PDPN/GD2-CAR T cell blend. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/636223v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@109a9aaorg.highwire.dtl.DTLVardef@16215faorg.highwire.dtl.DTLVardef@910623org.highwire.dtl.DTLVardef@129f79c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGRAPHICAL ABSTRACT:C_FLOATNO C_FIG

immunology↗

bioRxiv2025

Autocrine glucocorticoid signaling in hormonally active cancer induces antigen expression for immunotherapy

Exogenous glucocorticoids (GCs) suppress T cell-based immunotherapy, yet the consequences of endogenous GCs produced or regenerated by tumours remain poorly understood. Here, we show that tumour-derived GCs couple immune evasion to therapeutic antigenicity. In adrenocortical carcinoma, autonomous GC production activates hGR-STAT3 signaling to increase the surface antigen ROR1. This creates an immunological paradox in which GC excess promotes therapeutic antigen expression while suppressing T cell-mediated antitumor activity. Selective hGR-knockout renders ROR1 CAR-T cells GC-resistant while preserving GC-driven ROR1 expression in tumor cells, resulting in durable tumor control in vivo. Non-endocrine pancreatic and triple-negative breast cancers recreate this circuit through HSD11B1-mediated GC recycling, which is further induced by CAR-T cell-derived cytokines under immune pressure. Thus, GC-resistant CAR-T cells exploit a tumor-derived endocrine program that couples immune suppression to therapeutic antigenicity.

cancer biology↗

bioRxiv2024

Microbial metabolite-guided CAR T cell engineering enhances anti-tumor immunity via epigenetic-metabolic crosstalk

The microbiome is a complex host factor and key determinant of the outcome of antibody-based and cellular immunotherapy. Its postbiotics are a blend of soluble commensal byproducts that are released into the host environment and have been associated with the regulation of immune homeostasis, particularly through impacts on epigenetics and cell signaling. In this study, we show that the postbiotic pentanoate is metabolized to citrate within the TCA cycle via both the acetyl- and succinyl-CoA entry points, a feature uniquely enabled by the chemical structure of the C5 aliphatic chain. We identified ATP-citrate lyase as the crucial factor that redirects pentanoate-derived citrate from the succinyl-CoA route to the nucleus, thereby linking metabolic output and histone acetylation. This epigenetic-metabolic crosstalk mitigated T cell exhaustion and promoted naive-like differentiation in pentanoate-programmed chimeric antigen receptor (CAR) T cells. The predictive and therapeutic potential of pentanoate was corroborated in two independent patient cohorts and three syngeneic models of CAR T adoptive therapy. Our data demonstrate that postbiotics are integrated into mitochondrial metabolism and subsequently incorporated as epigenetic imprints. This bridge between microbial and mammalian interspecies communication can ultimately impact T cell differentiation and efficacy.

immunology↗

bioRxiv2023

CD56-mediated activation of human natural killer cells is triggered by Aspergillus fumigatus galactosaminogalactan

Invasive aspergillosis causes significant morbidity and mortality in immunocompromised patients. Natural killer (NK) cells are pivotal for antifungal defense. Thus far, CD56 is the only known pathogen recognition receptor on NK cells triggering potent antifungal activity against Aspergillus fumigatus. However, the underlying cellular mechanisms and the fungal ligand of CD56 have remained unknown. Using purified cell wall components, biochemical treatments, and A. fumigatus mutants with altered cell wall composition, we herein found that CD56 interacts with the A. fumigatus cell wall carbohydrate galactosaminogalactan (GAG). This interaction induced NK cell activation, degranulation, and secretion of immune-enhancing chemokines and cytotoxic effectors. Supernatants from GAG-stimulated NK cells elicited antifungal activity and enhanced antifungal effector responses of polymorphonuclear cells. In conclusion, we identified A. fumigatus GAG as a ligand of CD56 on human primary NK cells, stimulating potent antifungal effector responses and activating other immune cells.

microbiology↗

bioRxiv2023

CARs are organized in nanodomains in the plasma membrane of T cells that accumulate at tumor contact sites

Chimeric antigen receptors (CARs) are synthetic immune receptors that are expressed in T cells through genetic engineering. CAR-T cells have been successfully used to eradicate very advanced leukemias and lymphomas and their functional properties have been intensively studied. However, relatively little is known about the spatiotemporal expression and organization of CARs on the T-cell membrane and how this influences their efficacy. Here, we applied super-resolution microscopy to visualize CD19-, ROR1-, and ROR2-specific CARs in human CD4+ and CD8+ T cells that were engineered with lentiviral and transposon-mediated gene transfer. Our data show that the majority of CARs is organized in nanodomains virtually independent of the T cell type, CAR construct and expression level. Quantitative analyses revealed a slightly higher CAR density in transposon-engineered T cells correlating with higher antigen sensitivity and faster resolution of anti-tumor functions compared to lentivirally-engineered T cells. Live-cell fluorescence imaging revealed that both, CAR nanodomains and CAR monomers accumulate at tumor contact sites and form multifocal immunological synapses. Our study provides novel insights into the membrane organization of CARs with single-molecule resolution and illustrates the potential of advanced microscopy to inform the rational design of synthetic immune receptors for applications in immune cell therapy.

immunology↗

bioRxiv2019

Inefficient ZAP70-Signaling Blunts Antigen Detection by CAR-T-Cells

Rational design of chimeric antigen receptors (CARs) with optimized anti-cancer performance mandates detailed knowledge of how CARs engage tumor antigens and how antigen-engagement triggers activation. We analyzed CAR-mediated antigen recognition via quantitative single molecule live-cell imaging and found the sensitivity of CAR-T-cells towards antigen approximately 1000-times reduced when compared to T-cell antigen receptor (TCR)-mediated recognition of nominal peptide/MHC complexes. While CARs outperformed TCRs with regard to antigen binding within the immunological synapse, proximal signaling was significantly attenuated due to inefficient recruitment of the tyrosine-kinase ZAP70 to ligated CARs and its reduced concomitant activation and subsequent release. Our study exposes signaling deficiencies of state-of-the-art CAR-designs, which limit at present the efficacy of CAR-T-cell therapies to target tumors with diminished antigen expression.

immunology↗

bioRxiv2019

Genomic Analyses of SLAMF7 CAR-T Cells Manufactured by Sleeping Beauty Transposon Gene Transfer for Immunotherapy of Multiple Myeloma

Widespread treatment of human diseases with gene therapies necessitates the development of gene transfer vectors that integrate genetic information effectively, safely and economically. Accordingly, significant efforts have been devoted to engineer novel tools that i) achieve high-level stable gene transfer at low toxicity to the host cell; ii) induce low levels of genotoxicity and possess a safe integration profile with a high proportion of integrations into safe genomic locations; and iii) are associated with acceptable cost per treatment and scalable/exportable vector production to serve large numbers of patients. The Sleeping Beauty (SB) transposon has been transformed into a vector system that is fulfilling these requirements.\n\nIn the CARAMBA project, we use SB transposition to genetically modify T cells with a chimeric antigen receptor (CAR) specific for the SLAMF7 antigen, that is uniformly and highly expressed on malignant plasma cells in multiple myeloma. We have demonstrated that SLAMF7 CAR-T cells confer specific and very potent anti-myeloma reactivity in pre-clinical models, and are therefore preparing a Phase I/IIa clinical trial of adoptive immunotherapy with autologous, patient-derived SLAMF7-CAR T cells in multiple myeloma (EudraCT Nr. 2019-001264-30/CARAMBA-1).\n\nHere we report on the characterization of genomic safety attributes in SLAMF7 CAR-T cells that we prepared in three clinical-grade manufacturing campaigns under good manufacturing practice (GMP), using T cells that we obtained from three healthy donor volunteers. In the SLAMF7 CAR-T cell product, we determined the average transposon copy number, the genomic insertion profile, and presence of residual SB100X transposase. The data show that the SLAMF7 CAR transposon had been inserted into the T cell genome with the close-to-random distribution pattern that is typical for SB, and with an average transposon copy number ranging between 6 and 12 per T cell. No residual SB100X transposase could be detected by Western blotting in the infusion products. With these attributes, the SLAMF7 CAR-T products satisfy criteria set forth by competent regulatory authorities in order to justify administration of SLAMF7 CAR-T cells to humans in the context of a clinical trial. These data set the stage for the CARAMBA clinical trial, that will be the first in the European Union to use virus-free SB transposition for CAR-T engineering.\n\nDisclosuresThis project is receiving funding from the European Unions Horizon 2020 research and innovation programme under grant agreement No 754658 (CARAMBA).

bioengineering↗

bioRxiv2019

3D light sheet fluorescence microscopy of lungs to dissect local host immune - Aspergillus fumigatus interactions

Aspergillus fumigatus is an opportunistic fungal pathogen that can cause life-threatening invasive lung infections in immunodeficient patients. The cellular and molecular processes of infection during onset, establishment and progression are highly complex and depend on both fungal attributes and the immune status of the host. Therefore, preclinical animal models are paramount to investigate and gain better insight into the infection process. Yet, despite their extensive use, commonly employed murine models of invasive pulmonary aspergillosis are not well understood due to analytical limitations. Here we present quantitative light sheet fluorescence microscopy (LSFM) to describe fungal growth and the local immune response in whole lungs at cellular resolution within its anatomical context. We analyzed three very common murine models of pulmonary aspergillosis based on immunosuppression with corticosteroids, chemotherapy-induced leukopenia or myeloablative irradiation. LSFM uncovered distinct architectures of fungal growth and degrees of tissue invasion in each model. Furthermore, LSFM revealed the spatial distribution, interaction and activation of two key immune cell populations in antifungal defense: alveolar macrophages and polymorphonuclear neutrophils. Interestingly, the patterns of fungal growth correlated with the detected effects of the immunosuppressive regimens on the local immune cell populations. Moreover, LSFM demonstrates that the commonly used intranasal route of spore administration did not result in the desired intra-alveolar deposition, as more than 60% of fungal growth occurred outside of the alveolar space. Hence, LSFM allows for more rigorous characterization of murine models of invasive pulmonary aspergillosis and pinpointing their strengths and limitations.\n\nIMPORTANCEThe use of animal models of infection is essential to advance our understanding of complex host-pathogen interactions that take place during Aspergillus fumigatus lung infections. As in the case of humans, mice need to be immunosuppressed to become susceptible to invasive pulmonary aspergillosis, the most serious infection caused by A. fumigatus. There are several immunosuppressive regimens that are routinely used to investigate fungal growth and/or immune responses in murine models of invasive pulmonary aspergillosis (IPA). However, the precise consequences that each immunosuppressive model has on the local immune populations and for fungal growth are not completely understood. Here we employed light sheet fluorescence microscopy (LSFM) to analyze whole lungs at cellular resolution, to pin down the scenario commonly used IPA models. Our results will be valuable to optimize and refine animal models to maximize their use in future research.\n\nVISUAL ABSTRACTQuantitative light sheet fluorescence microscopy to dissect local host-pathogen interactions in the lung after A. fumigatus airway infection.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC=\"FIGDIR/small/661157v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (42K):\norg.highwire.dtl.DTLVardef@4baf31org.highwire.dtl.DTLVardef@dad3c3org.highwire.dtl.DTLVardef@84aab9org.highwire.dtl.DTLVardef@1e45c0d_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗