Biology subjects

Loeffler, J.

Publications and source records attributed to Loeffler, J..

6 recordsLinked to original sources

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

The membrane distal domain of CD16a allosterically regulates NK cell ADCC

Antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells is mediated by the activating IgG receptor CD16a (Fc{gamma}RIIIa), yet the molecular mechanisms governing receptor activation remain poorly understood. We demonstrate that the membrane-distal domain 1 (D1) of CD16a functions as an allosteric checkpoint that controls ADCC independently of IgG-Fc binding. A nanobody, C28, that binds an electronegative patch in D1 dose-dependently blocks NK cell ADCC against multiple therapeutic antibodies without affecting direct cytotoxicity. A second nanobody, C21, binding an adjacent D1 epitope has no such effect. Cryo-EM structures of the CD16a-IgG-nanobody complex reveal that C28 allosterically competes with core-fucosylated IgG and stabilizes a closed D1 conformation resembling unliganded receptor, even when Fc is bound. Molecular dynamics simulations show that occupation of the D1 epitope rigidifies the IgG-binding site, stabilizing CD16a overall in contrast with IgG binding alone. The nanobody C28 restricts CD3{zeta} phosphorylation in both resting and ADCC-activated NK cells, revealing tonic inhibitory control upstream of the signaling cascade. Using MINFLUX nanoscopy, we also show that CD16a forms dimers of [~]9 nm spacing on the NK cell surface, a geometry unaltered by the ADCC-enhancing L48H polymorphism. Drawing on structural parallels with the IgE receptor Fc{varepsilon}RI, which is held inactive as a cholesterol-stabilized dimer, we propose that CD16a dimerization through D1 contacts represents a conserved autoinhibitory mechanism among Fc receptors. Consistent with this model, structure-guided disruption of the C28 epitope in NK-92 cells enhances ADCC potency and killing kinetics, providing a blueprint for engineering improved cellular immunotherapeutics.

immunology↗

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

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↗

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↗

bioRxiv2019

Application of long-read sequencing for robust identification of correct alleles in genome edited animals

Recent developments in CRISPR/Cas9 genome editing tools have facilitated the introduction of more complex alleles, often spanning genetic intervals of several kilobases, directly into the embryo. These techniques often produce mosaic founder animals and the introduction of donor templates, via homologous directed repair, can be erroneous or incomplete. Newly generated alleles must be verified at the sequence level across the targeted locus. Screening for the presence of the desired mutant allele using traditional sequencing methods can be challenging due to the size of the desired edit(s) together with founder mosaicism. In order to help disentangle the genetic complexity of these animals, we tested the application of Oxford Nanopore long read sequencing of the targeted locus. Taking advantage of sequencing the entire length of the segment in each single read, we were able to determine whether the entire intended mutant sequence was present in both mosaic founders and their offspring.

molecular biology↗