Jochen Behrends

Non-specific (ns) lipid transfer proteins (LTPs) are lipid-binding allergens whose natural ligands are not fully known. To elucidate the function and allergenic relevance of nsLTP-lipid complexes, purified natural Lupinus luteus ( L. luteus ) nLTP and recombinant peach LTP, rPru p 3, were tested for membrane interaction and lipid transport activity using liposome assays and Förster-resonance-energy-transfer (FRET) in a case-level proof-of-principle investigation. Allergenic relevance of the LTP-lipid interaction was evaluated in the presence of oleic acid (OA), phosphatidylcholine (PC), phosphatidylglycerol (PG), and phosphatidylserine (PS) in a basophil activation test (BAT) with effector cells from an LTP-allergic patient. Both LTPs interacted with neutral PC and negatively charged PS liposomal membranes. A novel transport activity for anionic PG species was identified for both proteins, indicating a shared functional preference for the glycerol headgroup. LTP-dependent lipid exchange/mixing were consistent with transfer. However, fusion/mixing mechanisms cannot be excluded with the current readout. In BAT, both LTPs showed enhanced activation in combination with OA, PC, PG, and PS. As PG is a key component of bacterial membranes, the PG specificity of the lipid interaction of L. luteus nLTP and rPru p 3 is likely of relevance in allergen interaction with the gut microbiome and for enhancement of allergic symptoms. These findings highlight lipid-specific functional properties and lipid-dependent modulation of allergenic activity in plant nsLTPs.

Selective enrichment of extracellular vesicle (EV) subpopulations from the heterogeneous EV pool is essential for understanding their characteristic biological functions and exploiting their potential as diagnostic and prognostic biomarkers. However, isolation of specific EV-subsets remains challenging. Fluorescence-Activated Cell Sorting (FACS) has emerged as promising technique for EV subpopulations enrichment, despite limitations associated to their small size. Although FACS-based EV sorting has been reported, a broadly applicable and systematically validated workflow is still lacking. Here, we describe and validate an optimized workflow for the sorting and analysis of EVs derived from diverse species, tissues, blood and cell culture systems. Using two advanced flow cytometric cell sorters, the BD FACSAria Fusion, and the BD FACSDiscover S8, we systematically evaluated key technical parameters, including nozzle size, sample dilutions, and sorting mode. The optimized workflow enabled efficient enrichment of differently labelled EV populations of interest, achieving near-100% purity, including rare subsets representing less than 10% of the total EV pool, while maintaining compatibility with downstream analyses. Sorted EV populations were characterized by high-sensitivity imaging flow cytometry, transmission electron microscopy, and liquid chromatography–tandem mass spectrometry. This workflow provides a robust framework for EV subset isolation and characterization, supporting both fundamental EV research and translational biomarker applications.

Tuberculosis (TB) remains a leading global cause of death, with approximately 1.3 million fatalities annually. While males are more susceptible to TB, the underlying immune differences and their impact on vaccine efficacy are not fully understood. In this study, we vaccinated male and female C57BL/6 mice with BCG and 2 recombinant derivatives, VPM1002 and BCGΔBCG1419c, and assessed their protection against Mycobacterium tuberculosis (Mtb) HN878. While all 3 vaccines showed comparable efficacy in females, BCG did not protect males from Mtb-induced death. Instead, recombinant vaccines provided improved protection in males. Vaccination of males with BCGΔBCG1419c, but not BCG, enhanced CD8 T-cell and B-cell recall responses in vaccine-draining lymph nodes, and reduced lung inflammation after Mtb challenge. These findings underscore the impact of sex differences on vaccine efficacy and suggest that future research should consider sex as a biological variable to optimize TB vaccination strategies.

Background: Virus infections drive COPD exacerbations and progression. Antiviral immunity centres on the activation of virus-specific CD8⁺T-cells by viral epitopes presented on major histocompatibility complex (MHC) class I molecules of infected cells. These epitopes are generated by the immunoproteasome, a specialised intracellular protein degradation machine, which is induced by antiviral cytokines in infected cells.Methods: We analysed the effects of cigarette smoke on cytokine- and virus-mediated induction of the immunoproteasomein vitro,ex vivoandin vivousing RNA and Western blot analyses. CD8⁺T-cell activation was determined in co-culture assays with cigarette smoke-exposed influenza A virus (IAV)-infected cells. Mass-spectrometry-based analysis of MHC class I-bound peptides uncovered the effects of cigarette smoke on inflammatory antigen presentation in lung cells. IAV-specific CD8⁺T-cell numbers were determined in patients’ peripheral blood using tetramer technology.Results: Cigarette smoke impaired the induction of the immunoproteasome by cytokine signalling and viral infection in lung cellsin vitro,ex vivoandin vivo. In addition, cigarette smoke altered the peptide repertoire of antigens presented on MHC class I molecules under inflammatory conditions. Importantly, MHC class I-mediated activation of IAV-specific CD8⁺T-cells was dampened by cigarette smoke. COPD patients exhibited reduced numbers of circulating IAV-specific CD8⁺T-cells compared to healthy controls and asthmatics.Conclusion: Our data indicate that cigarette smoke interferes with MHC class I antigen generation and presentation and thereby contributes to impaired activation of CD8⁺T-cells upon virus infection. This adds important mechanistic insight on how cigarette smoke mediates increased susceptibility of smokers and COPD patients to viral infections.

During experimental tuberculosis (TB), interleukin (IL)-17A appears to be involved in the formation of lung granulomas, possibly through the attraction of neutrophils to the sites of infection. However, the protective impact of cytokine appears to depend on the degree of its induction. Hence, robust production of IL-17A in mice infected with the hypervirulent isolate Mycobacterium tuberculosis (Mtb) HN878 mediates protection, while the cytokine is dispensable for protective immune responses against low-dose infection with the less virulent strain H37rv. Here, we show that after experimental infection with high doses of Mtb H37rv, IL-17A-deficient (^−/−) mice exhibited high susceptibility to the infection, which was mediated by the strong accumulation of neutrophils in the infected lung tissue. Accordingly, we observed nearly unrestricted bacterial replication within the neutrophils, indicating that they may serve as a survival niche for Mtb. By use of IL-17A/IL-17F-double-deficient mice, we demonstrated that the susceptibility in the absence of IL-17A is mediated by a compensatory expression of IL-17F, which, however, appeared not to be dependent on neutrophils. Together, our results illustrate the compensatory potential of the Th17-secreted cytokines IL-17A and IL-17F in the context of experimental TB and once again emphasize the detrimental effect of excessive neutrophil infiltration in response to Mtb.

Immune cells at sites of inflammation are continuously activated by local antigens and cytokines, and regulatory mechanisms must be enacted to control inflammation. The stepwise hydrolysis of extracellular ATP by ectonucleotidases CD39 and CD73 generates adenosine, a potent immune suppressor. Here we report that human effector CD8 T cells contribute to adenosine production by releasing CD73-containing extracellular vesicles upon activation. These extracellular vesicles have AMPase activity, and the resulting adenosine mediates immune suppression independently of regulatory T cells. In addition, we show that extracellular vesicles isolated from the synovial fluid of patients with juvenile idiopathic arthritis contribute to T cell suppression in a CD73-dependent manner. Our results suggest that the generation of adenosine upon T cell activation is an intrinsic mechanism of human effector T cells that complements regulatory T cell-mediated suppression in the inflamed tissue. Finally, our data underscore the role of immune cell-derived extracellular vesicles in the control of immune responses.

Influenza during pregnancy can affect the health of offspring in later life, among which neurocognitive disorders are among the best described. Here, we investigate whether maternal influenza infection has adverse effects on immune responses in offspring. We establish a two-hit mouse model to study the effect of maternal influenza A virus infection (first hit) on vulnerability of offspring to heterologous infections (second hit) in later life. Offspring born to influenza A virus infected mothers are stunted in growth and more vulnerable to heterologous infections (influenza B virus and MRSA) than those born to PBS- or poly(I:C)-treated mothers. Enhanced vulnerability to infection in neonates is associated with reduced haematopoetic development and immune responses. In particular, alveolar macrophages of offspring exposed to maternal influenza have reduced capacity to clear second hit pathogens. This impaired pathogen clearance is partially reversed by adoptive transfer of alveolar macrophages from healthy offspring born to uninfected dams. These findings suggest that maternal influenza infection may impair immune ontogeny and increase susceptibility to early life infections of offspring.

In view of emerging drug-resistant tuberculosis (TB), host-directed adjunct therapies are urgently needed to improve treatment outcomes with currently available anti-TB therapies. One approach is to interfere with the formation of lipid-laden “foamy” macrophages in the host, as they provide a nutrient-rich host cell environment for Mycobacterium tuberculosis (Mtb). Here, we provide evidence that Wnt family member 6 (WNT6), a ligand of the evolutionarily conserved Wingless/Integrase 1 (WNT) signaling pathway, promotes foam cell formation by regulating key lipid metabolic genes including acetyl-CoA carboxylase 2 (ACC2) during pulmonary TB. Using genetic and pharmacological approaches, we demonstrated that lack of functional WNT6 or ACC2 significantly reduced intracellular triacylglycerol (TAG) levels and Mtb survival in macrophages. Moreover, treatment of Mtb-infected mice with a combination of a pharmacological ACC2 inhibitor and the anti-TB drug isoniazid (INH) reduced lung TAG and cytokine levels, as well as lung weights, compared with treatment with INH alone. This combination also reduced Mtb bacterial numbers and the size of mononuclear cell infiltrates in livers of infected mice. In summary, our findings demonstrate that Mtb exploits WNT6/ACC2-induced storage of TAGs in macrophages to facilitate its intracellular survival, a finding that opens new perspectives for host-directed adjunctive treatment of pulmonary TB.

Background: Allergy is one of the most common chronic diseases in Europe. Therefore, an increased need for specific and sensitive diagnostic tests that truly detect allergy exists. This study aimed at establishing a highly specific high‐throughput and automated basophil activation test (BAT) that proves the existence of an allergy with utmost probability.Methods: BAT from 1104 samples was analyzed; a novel gating strategy with three antibodies (FcεRIα, CD203c, CD63) was established and compared with our published protocol (12 antibodies). Based on the novel gating strategy, storage conditions, automated measurement, and analyses using R (1376 samples out of 1389) were optimized to set up a high‐throughput BAT.Results: No differences in sensitivity and specificity were found between the novel three antibody (FcεRIα, CD203c, CD63) and the 12 antibody gating strategy or between automated and manually analyzed samples (saving up to 90% of labor time). The time frame for basophil activation measurement after blood donation has been extended considerably (whole blood storage ≤7 days (RT) and 17 days (4°C) prior to BAT preparation and measurement). Respective storage conditions were optimized for samples after stimulation, staining, and preparation (≤7 days (RT) and 28 days (4°C)). These achievements were confirmed by a nationwide ring trial showing robustness and applicability of our BAT on a variety of flow cytometers.Conclusion: Our considerable optimizations overcame the hurdles that until now prevented the BAT from being used as high‐throughput allergy diagnostic test in routine laboratories and shall allow for collaborative studies between clinics and research centers.