Anne Krug

Maintaining a balanced immunity between pathogen defense and tolerance to environmental antigens in neonates is essential for survival and the establishment of life-long immune homeostasis. Instructed by environmental signals, type 1 conventional dendritic cells (cDC1) contribute to both processes but how the balance may be achieved is unclear. Here, we uncover an interferon (IFN)γ-driven regulatory circuit in early life that relays dietary cues to spleen cDC1. IFNγ-mediated STAT1-signaling induces an immunogenic maturation program in spleen cDC1 that enables them to shape the effector differentiation of antigen-experienced effector memory CD8⁺ T cells. This cDC1 program emerges during the transition from breastfeeding to solid food at weaning, occurs in germ-free mice, and remains operative to dietary intervention in adult mice. At weaning, this IFNγ signal enables spleen cDC1 to shape the effector phenotype of food-antigen-specific CD8 ⁺ T cells in a feedforward manner, thereby recalibrating the developing T cell pool. Our findings identify diet as a modifiable cue that can tune systemic cDC1-mediated immunity, opening new opportunities to steer immune responses during early life and beyond.

The live-attenuated yellow fever vaccine 17D-204 (YF17D) activates robust innate immune responses followed by rapid induction of adaptive immunity resulting in long-lasting protection. YF17D triggers the production of type I interferons (IFNs) which have a dual role in antigen presenting cells regulating their infection and contributing to their activation. Infection with YF17D was detected in primary human blood monocytes and conventional dendritic cells (DCs) and in monocyte-derived DCs but was highly restricted by type I IFN. Blocking IFNAR signaling in YF17D-infected PBMC from vaccinated donors resulted in increased activation of YF17D-specific CD8 ⁺ T cells. Consistently, peak IFN-alpha plasma levels correlated inversely with the CD8 ⁺ T cells response in YF17D vaccinees. Loss of function experiments demonstrated a dominant role of retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) and mitochondrial antiviral signaling protein (MAVS) for type I IFN induction and restriction of YF17D. The type I IFN response was mediated by 5’ tri- or diphosphate dsRNA intermediates that are formed during YF17D infection. In vivo proximity labelling (IPL) of RIG-I and next-generation sequencing confirmed interaction of RIG-I with YF17D-dsRNA in infected cells. Thus, YF17D-triggered RLR-signaling restricts viral replication through type I IFN and thus limits the production of viral antigens that can be presented to T cells.

Yellow fever vaccination provides long-lasting protection and is a unique model for studying the immune response to an acute RNA virus infection in humans. To elucidate the early innate immune events preceding the rapid generation of protective immunity, we performed transcriptome analysis of human blood dendritic cell (DC) and monocyte subpopulations before and 3, 7, 14, and 28 d after vaccination. We detected temporary upregulation of IFN-stimulated genes (ISG) in all DC and monocyte subsets on days 3 and 7 after vaccination as well as cell type–specific responses and response kinetics. Single-cell RNA sequencing revealed rapid appearance of activated DC and monocyte clusters dominated by ISGs, inflammatory chemokines, and genes involved in antigen processing and presentation. This was confirmed by flow cytometric analysis in a large cohort of vaccinees. We identified SIGLEC1/CD169 upregulation as a sensitive indicator of the transient IFN-induced activation state elicited in DCs and monocytes by YF17D vaccination correlating with early protective IgM antibody responses.

Phosphatidylserine (PS) is a well-established marker for apoptotic cells and activated platelets, and it is also found on extracellular vesicles (EVs). However, the frequency of PS ⁺ EVs remains a topic of debate, probably due to the distinct lipid composition of EVs, which varies with the source material. This variation underscores the need for highly sensitive detection reagents to accurately differentiate between PS ⁺ and PS ⁻ EVs. To address this issue, we compared several PS-binding reagents for the staining of blood EVs, including milk fat globule factor E8 (MFG-E8, lactadherin), a derivative of MFG-E8, and Annexin V, the most used compound in the literature. Here, we demonstrate the predominance of PS ⁺ EVs in both murine and human blood and reveal significant differences in the affinities of the PS-binding proteins. With approximately 90% of the detected blood EVs being PS ⁺ , we utilized a derivative of MFG-E8 for in vivo monitoring of both circulating and cell-bound EVs, providing a novel tool to analyze endogenous EV kinetics. Our findings showed that murine PS ⁺ plasma EVs were rapidly cleared from circulation (∼50% after 30 minutes), yet they remained present on splenic B cells and monocytes/macrophages. In conclusion, MFG-E8-based reagents enable externalized PS to serve as a universal EV marker, greatly improving EV isolation, standardizing EV quantification, and facilitating diagnostic EV monitoring and disease biomarker discovery.

Yellow fever vaccination provides long-lasting protection and is a unique model for studying the immune response to an acute RNA virus infection in humans. To elucidate the early innate immune events preceding the rapid generation of protective immunity, we performed transcriptome analysis of human blood dendritic cell (DC) and monocyte subpopulations before and 3, 7, 14 and 28 days after vaccination. We detected temporary upregulation of IFN-stimulated genes (ISG) in all DC and monocyte subsets on day 3 and 7 after vaccination as well as cell type specific responses and temporal dynamics. Single cell RNA sequencing revealed rapid appearance of activated DC and monocyte clusters dominated by ISGs, inflammatory chemokines and genes involved in antigen processing and presentation. This was confirmed by flow cytometric analysis in a large cohort of vaccinees. We identified SIGLEC1/CD169 upregulation as a sensitive indicator of the transient IFN-induced activation state elicited in DCs and monocytes by YF17D vaccination correlating with early protective IgM antibody responses.

Cells sense chemical and mechanical signals within their local microenvironment. To sample for chemical signals, cells employ bulk extracellular fluid uptake through macropinocytosis in a receptor-independent manner. Although macropinocytosis occurs in microenvironments of multicellular organisms, it remains largely unknown whether and how the microenvironment itself regulates fluid sampling. Here, we reveal that the microenvironmental properties regulate fluid sampling by macropinocytosis. Using macrophages as constitutively sampling immune cells, we discover that macropinocytosis in three-dimensional (3D) microenvironments is more efficient than on flat 2D surfaces. Enhanced macropinocytosis in 3D is driven by low adhesiveness to the cellular substrate, releasing the mechanical linkage between the cell and its surroundings, thereby facilitating the formation of actin-based membrane protrusions that sample the local substrate-free space. Mechanistically, we identify that the microenvironment regulates the utilization of macropinocytosis-competent actin networks, as the actin nucleating Arp2/3 complex and its regulator Hem1 particularly drive macropinocytosis in 3D. Our results establish microenvironmental properties as stimulators and regulators of macropinocytosis, providing important implications for fluid sampling during physiology, immunity, and diseases. Additionally, our findings identify a reciprocal relationship between the microenvironment and fluid sampling, wherein cells acquire information from the microenvironment by macropinocytosis, while this process, in turn, is regulated by the microenvironment.

The yellow fever 17D vaccine (YF17D) is highly effective but is frequently administered to individuals with pre-existing cross-reactive immunity, potentially impacting their immune responses. Here, we investigate the impact of pre-existing flavivirus immunity induced by the tick-borne encephalitis virus (TBEV) vaccine on the response to YF17D vaccination in 250 individuals up to 28 days post-vaccination (pv) and 22 individuals sampled one-year pv. Our findings indicate that previous TBEV vaccination does not affect the early IgM-driven neutralizing response to YF17D. However, pre-vaccination sera enhance YF17D virus infection in vitro via antibody-dependent enhancement (ADE). Following YF17D vaccination, TBEV-pre-vaccinated individuals develop high amounts of cross-reactive IgG antibodies with poor neutralizing capacity. In contrast, TBEV-unvaccinated individuals elicit a non-cross-reacting neutralizing response. Using YF17D envelope protein mutants displaying different epitopes, we identify quaternary dimeric epitopes as the primary target of neutralizing antibodies. Additionally, TBEV-pre-vaccination skews the IgG response towards the pan-flavivirus fusion loop epitope (FLE), capable of mediating ADE of dengue and Zika virus infections in vitro. Together, we propose that YF17D vaccination conceals the FLE in individuals without prior flavivirus exposure but favors a cross-reactive IgG response in TBEV-pre-vaccinated recipients directed to the FLE with potential to enhance dengue virus infection.

The immune system is in place to assist in ensuring tissue homeostasis, which can be easily perturbed by invading pathogens or nonpathogenic stressors causing tissue damage. Extracellular nucleotides are well known to contribute to innate immune signaling specificity and strength, but how their signaling is relayed downstream of cell surface receptors and how this translates into antiviral immunity is only partially understood. Here, we systematically investigated the responses of human macrophages to extracellular nucleotides, focusing on the nucleotide‐sensing GPRC receptors of the P2Y family. Time‐resolved transcriptomic analysis showed that adenine‐ and uridine‐based nucleotides induce a specific, immediate, and transient cytokine response through the MAPK signaling pathway that regulates transcriptional activation by AP‐1. Using receptor trans‐complementation, we identified a subset of P2Ys (P2Y1, P2Y2, P2Y6, and P2Y11) that govern inflammatory responses via cytokine induction, while others (P2Y4, P2Y11, P2Y12, P2Y13, and P2Y14) directly induce antiviral responses. Notably, P2Y11 combined both activities, and depletion or inhibition of this receptor in macrophages impaired both inflammatory and antiviral responses. Collectively, these results highlight the underappreciated functions of P2Y receptors in innate immune processes.

Human pathogenic flaviviruses pose a significant health concern and vaccination is the most effective instrument to control their circulation. How pre-existing immunity to antigenically related viruses modulates immunization outcome remains poorly understood. In this study, we evaluated the effect of vaccination against tick-borne encephalitis virus (TBEV) on the epitope immunodominance and immunogenicity of the yellow fever 17D vaccine (YF17D) in a cohort of 250 human vaccinees. Following YF17D vaccination, all study participants seroconverted and generated protective neutralizing antibody titers. At day 28, TBEV pre-immunity did not affect the polyclonal neutralizing response which largely depended on the IgM fraction. We found that sera from TBEV-immunized individuals enhanced YF17D vaccine virus infection via antibody-dependent enhancement (ADE). Upon vaccination, individuals with TBEV pre-immunity had higher concentrations of cross-reactive IgG antibodies with limited neutralizing capacity against YF17D whereas vaccinees without prior flavivirus exposure showed a non-cross-reacting response. Using a set of recombinant YF17D envelope protein mutants displaying different epitopes, we identified quaternary epitopes as the primary target of neutralizing antibodies. Sequential immunizations redirected the IgG response towards the pan-flavivirus fusion loop epitope (FLE) with the potential to mediate enhancement of dengue and Zika virus infections whereas TBEV naïve individuals elicited an IgG response directed towards neutralizing epitopes without an enhancing effect. We propose that the YF17D vaccine effectively conceals the FLE and primes a neutralizing IgG response in individuals with no prior flavivirus exposure. In contrast, the response in TBEV-experienced recipients favors weakly-neutralizing, cross-reactive epitopes potentially increasing the risk of severe dengue and Zika disease due to ADE.

Dysregulation of the myeloid cell compartment is a feature of severe disease in hospitalized COVID‐19 patients. Here, we investigated the response of circulating dendritic cell (DC) and monocyte subpopulations in SARS‐CoV‐2 infected outpatients with mild disease and compared it to the response of healthy individuals to yellow fever vaccine virus YF17D as a model of a well‐coordinated response to viral infection. In SARS‐CoV‐2‐infected outpatients circulating DCs were persistently reduced for several weeks whereas after YF17D vaccination DC numbers were decreased temporarily and rapidly replenished by increased proliferation until 14 days after vaccination. The majority of COVID‐19 outpatients showed high expression of CD86 and PD‐L1 in monocytes and DCs early on, resembling the dynamic after YF17D vaccination. In a subgroup of patients, low CD86 and high PD‐L1 expression were detected in monocytes and DCs coinciding with symptoms, higher age, and lower lymphocyte counts. This phenotype was similar to that observed in severely ill COVID‐19 patients, but less pronounced. Thus, prolonged reduction and dysregulated activation of blood DCs and monocytes were seen in a subgroup of symptomatic non‐hospitalized COVID‐19 patients while a transient coordinated activation was characteristic for the majority of patients with mild COVID‐19 and the response to YF17D vaccination.