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.

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.

Separating and enriching specific extracellular vesicle (EV) subpopulations from the broader EV pool present in tissues and blood is crucial for understanding their role in physiological and pathological conditions. However, high-purity enrichment of specific EV-subpopulations remains challenging due to the lack of suitable techniques. Initial studies have shown that Fluorescence-Activated Cell Sorting (FACS) has great potential for enriching EV subpopulations, despite the technical challenges posed by their small size. Yet, existing protocols have been inconsistent, and proper validation using state-of-the-art sorters has been inadequate. Here, we introduce an EV sorting workflow that overcomes technical challenges and allows for the analysis of EVs from various species, tissue sources and cell culture. We used two fluorescence cell sorters, the BD FACSAria Fusion and the BD FACSDiscover S8, to sort EVs with different fluorescent labels. The successful sorting of EVs was validated using high-sensitivity imaging flow cytometry, transmission electron microscopy, and liquid chromatography tandem mass spectrometry. We defined the optimal parameters for nozzle sizes, flow rates, sample dilutions, and sorting modes, enabling the enrichment of EV populations of interest to nearby 100% purity, including low-frequency EV populations of under 10%, while preserving compatibility with downstream analyses. The workflow presented here provides a powerful tool for both, basic science and translational applications. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64 German Center for Child and Adolescent Health Schilling Foundation T. Von Zastrow Foundation Fielmann Foundation Werner-Otto Stiftung Else Kröner-Fresenius-Stiftung Jung-Stiftung für Wissenschaft und Forschung Studienstiftung des deutschen Volkes

BackgroundIn addition to serving as the primary effector cells against infections, neutrophils have been implicated in the regulation of both innate and adaptive immunity. In this study, we aimed to investigate the role of neutrophils in the regulation of the immune system under physiological conditions.MethodsThe in vivo effect of neutrophils on the immune system was examined using neutropenic mice. The interaction between neutrophils and γδ T cells was investigated using an in vitro co-culture system.FindingsUnexpectedly, we observed an accumulation of γδ T cells in the cervical lymph nodes of neutropenic mice. Transcriptomic analysis revealed that these γδ T cells exhibited unique expression profiles of cell surface molecules and genes involved in defense responses. Further characterization indicated that the accumulated γδ T cells were IL-17 producing CD44+CD62L−CD27− memory cells. Additionally, in vitro experiments demonstrated that neutrophils could inhibit the function of IL-17A producing γδ T cells by inducing cell death in a contact-dependent manner.ConclusionThis present study demonstrates that neutrophils negatively regulate IL-17 producing γδ T cells under physiological conditions. Given that IL-17A is a critical cytokine for the recruitment of neutrophils to peripheral tissues, our study suggests that the crosstalk between neutrophils and IL-17A producing γδ T cells is a crucial mechanism for maintaining immune homeostasis under physiological conditions.

IntroductionDue to the historical dogma that host defense against intracellular pathogens is primarily mediated by cell-mediated immunity, B cells have long been considered unimportant in providing protection against Mycobacterium tuberculosis (Mtb) and remained understudied for decades. However, emerging evidence highlights the multifaceted role of B cells in tuberculosis (TB) immunity. B cells accumulate at the site of infection in both animal models and human TB patients, suggesting a potential link to protective immunity. Still, the diverse roles of B cells in TB immunity are still being unraveled. In addition to producing antibodies, B cells secrete a wide range of cytokines that can influence the local immune response. In this study, we focused on the relevance of interleukin 10 (IL-10)-secreting B cells in the long-term control of the Mtb Beijing strain HN878.MethodsB cell-specific IL-10 expression was assessed in IL-10 transcriptional reporter (Vert-X) mice following Mtb infection. To investigate the role of B cell-derived IL-10 in TB immunity, both male and female mice with a targeted knockout of IL-10 in B cells (IL-10flox/CD19cre) were infected with Mtb HN878. Disease progression, control of bacterial replication, and immunological changes were monitored throughout the course of infection.ResultsB cells contribute to IL-10 production in the Mtb-infected lung in both sexes, with CD138+ plasma cells serving as the primary source of B cell-derived IL-10. Mice lacking B cell-derived IL-10 exhibited increased resistance to aerosol Mtb infection, demonstrated by a delayed onset of clinical symptoms and prolonged survival. Notably, this effect was significantly more pronounced in males compared to females, and was associated with male-specific immune alterations.ConclusionOur research highlights a previously unrecognized sex-specific regulatory role of B cell-derived IL-10 during Mtb infection.

Due to the historical dogma, that host defense against intracellular pathogens is mediated by cell-mediated immunity, B cells have been considered unimportant in providing protection against Mycobacterium tuberculosis ( Mtb ) and remained understudied for decades. However, emerging evidence suggests a more complex and multifaceted role for B cells in tuberculosis (TB) immunity. They accumulate at the side of infection in both animal models and human TB patients, suggesting a potential link to protective immunity. Still, the diverse roles of B cells in TB immunity continue to be unraveled. Apart from antibodies, B cells produce a wide range of cytokines, which can influence the local immune response. Here we addressed the relevance of interleukin 10 (IL-10) secreting B cells in long-term control of the Mtb Beijing strain HN878. Our research highlights the previously unknown role of B cell-derived IL-10 as a negative regulator of protective immunity in TB. For the first time, we demonstrate that mice lacking B cell-derived IL-10 show increased resistance to aerosol Mtb infection, as evidenced by a delayed onset of clinical symptoms and prolonged survival. Notably, this effect was significantly more pronounced in males compared to females, and was accompanied by male-specific immune alterations, indicating a previously unknown sex-specific regulatory role of B cell-derived IL-10 during Mtb infection. ### Competing Interest Statement The authors have declared no competing interest.

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 immunoproteasome in vitro, ex vivo and in vivo using 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 cells in vitro, ex vivo and in 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.

Tuberculosis (TB) is a disease that has evolved with humankind for millennia, causing approximately 1.3 million deaths worldwide per annum. Although increased male affliction for TB and other infections were long known from an epidemiological perspective, our mechanistic understanding of the underlying immunological divergences is relatively recent. As such, there is insufficient knowledge regarding the sexually dimorphic immune response to TB vaccines, where no accepted correlates of protection are yet available. In this context, our goal was to explore how individual sex influences the protective effects of TB vaccines. For this purpose, we vaccinated female and male C57BL/6 mice with Bacille Calmette-Guerin (BCG) and two recombinant derivatives, VPM1002 and BCGΔBCG1419c, to analyse their protective efficacy against challenge with Mycobacterium tuberculosis HN878. We found poor efficacy of BCG in males and the ability of next generation vaccine candidates to improve protection specifically in males. To determine the underlying mechanisms for the differences in survival upon vaccination between females and males, as well as, among different vaccine candidates, we analysed the distribution and persistence of the vaccine strains, in addition to vaccine-induced immune responses at various time points in draining lymph nodes and spleen. We identified sex specific differences in CD8 T cell proliferation in response to mycobacterial antigens ex vivo, 90 days post-vaccination, that associates with vaccine mediated protection against HN878. By integrating our multi-parametric datasets into principal component analysis, followed by extraction of high-variance features, we have uncovered an additional significant association of early CD4 T cell responses with late CD8 T cell responses as well as with survival post HN878 infection. In addition, we have also identified specific clusters of responding CD8 T cells in spleen post-vaccination, that are globally deficient in males as compared to females, irrespective of the BCG strain administered.

To the Editor, Immunoglobulin E (IgE) plays a crucial role in allergic reactions, including systemic anaphylaxis, by binding to the highaffinity IgE receptor FcεRI (FcεRIα + FcεRIβ + FcεRIγ) on mast cells and basophils and, upon allergenmediated aggregation, inducing the release of inflammatory mediators.1– 3 The allergic potential of IgE antibodies (Abs) is further affected by IgE glycosylationdependent interactions with membranebound and soluble carbohydratebinding proteins (e.g., galectins, siglecs, and Ctype lectin receptors) as well as different structural features (E14).1,3 Many healthy individuals harbor allergenspecific IgE Abs in the absence of an allergic disease (E5, E6), suggesting qualitative differences in IgEtriggered responses. Similarly, not all allergic individuals develop severe disease forms (e.g., asthma), and it is still unclear why allergen immunotherapy (AIT) is effective in some individuals but not in all (E6). Different natural or AITinduced inflammatory states of the specific Tand Bcell response may lead, as described for IgG (E7),4 to differently glycosylated IgE Abs that contribute distinctly to the induction of allergic responses. IgE Abs have several conserved Nglycosylation sites in the constant region of their heavy chains. Most of them are of the complex type and potentially galactosylated and terminal sialylated2,5 (Figure 1A,B). By comparing the effects of sialylated versus enzymatically desialylated IgE monoclonal (m)Abs, a recent study suggested that highly sialylated IgE Abs have a stronger potential to induce allergic reactions than lower sialylated IgE Abs.6 Notably, this observation is opposite to the findings that sialylation dampens the inflammatory potential of IgG, IgA, and IgM Abs (E8E10).4,7 Here, we further investigated the role of IgE sialylation on FcεRIα interaction, mast cell and basophil loading and activation, and IgEmediated passive systemic anaphylaxis (PSA) in mice. Therefore, we generated an antiTNP (2,4,6trinitrophenol) murine IgE mAb with three different sialylation levels: (i) originally sialylated (originally sial; glycosylation form generated by the hybridoma cell line), (ii) enzymatically desialylated (desial), and (iii) enzymatically additional sialylated (high sial) (Figure 1B– D). Both enzymatic IgE glycan modifications did not influence antigen binding (Figure 1E). Desialylated antiTNP IgE mAbs showed a stronger interaction with human and murine FcεRIα than originally sialylated and additionally sialylated IgE mAbs, whereas the latter two sialylated IgE glycoforms showed a comparable low or no interaction with FcεRIα in these assays, respectively (Figure 1F). This observation was unexpected due to the mentioned findings about the lower allergic potential of desialylated compared to sialylated IgE Abs6 but is in line with a stronger interaction of desialylated IgG Abs with Fcγ receptors than sialylated IgG Abs (E8). A comparable trend was observed when human mast cells differentiated from CD34+ blood cells were activated with the three antiTNP IgE glycoforms and TNPOVA as antigen (Figure S1). Moreover, less sialylated antiTNP IgE mAbs showed an enhanced loading on the human mast cell line LAD2 as well as on human blood basophils, which correlated positively with their activation (Figure 1G– J and Figure S1). Low sensitivity of the FcεRIα interaction assay and of the differentiated human mast cell assay and/or additional factors to FcεRIα binding might explain the higher loading and activation potentials of originally sialylated (orig. sial) compared to additional sialylated (high sial) IgE Abs in the LAD2 mast cell and blood basophil assays. Together, these data suggest that the higher interaction of de−/less sialylated IgE with mast cells and basophils correlate with their stronger activation potential. Next, we compared the potential of the three antiTNP IgE mAb glycoforms in an IgEmediated passive systemic anaphylaxis (PSA) mouse model (Figure 2A and Figure S1). In accordance with the data described by Shade et al., 20206 but in contrast to the expectation based on our in vitro data, intravenous (i.v.) injection of desialylated antiTNP IgE mAbs, followed by i.v. injection of TNPcoupled ovalbumin (TNPOVA) as “allergen” 6 or 24 h later, hardly induced any anaphylaxis, as measured by the drop in body core/rectal temperature, compared to the sialylated IgE mAbs (Figure 2B and Figure S1). However, we realized that desialylated antiTNP IgE mAbs could neither be detected in serum nor on blood basophils already 6 h after injection (Figure 2C,D and Figure S1). These data suggested a highly reduced serum halflife of desialylated IgE mAbs, which has also been described for desialylated IgA Abs in an asialoglycoprotein receptordependent manner (E11). To counteract the potential quick clearance of desialylated IgE mAbs, we blocked asialoglycoprotein receptors with an excess of desialylated (asialo)α1acid glycoprotein (AGP; orosomucoid) (E11) before sensitizing mice with the antiTNP IgE glycoforms (Figure 2E

Non-specific lipid transfer proteins (nsLTPs) as the primary sensitizer in plant-food allergic patients used to be seen primarily in the Mediterranean area. However, more recently, increasing numbers of clinically relevant sensitizations are being observed in Northern Europe. We herein report an unusual case of a woman who developed an anaphylactic reaction during a meal including a variety of different foods ranging from fruits and nuts to oats, wheat, and salmon. Allergy diagnostics showed no Bet v 1 sensitization but an nsLTP-mediated food allergy. Despite the much more prominent birch food syndrome in Central and Northern Europe, LTPs should be considered disease-causing agents, especially for patients developing severe reactions after consuming LTP-containing foods.

Progression of prostate cancer (PCa) is characterized by metastasis and castration resistance after response to androgen deprivation. Therapeutic options are limited, causing high morbidity and lethality. Recent work reported pro-oncogenic implications of the Mediator subunits cyclin-dependent kinase (CDK) 8 and 19 for the progression of PCa. The current study explored the underlying molecular mechanisms of CDK8/CDK19 and tested effects of novel CDK8/CDK19 inhibitors. PC3, DU145, LNCaP, and androgen-independent LNCaP Abl were used for in vitro experiments. Two inhibitors and CDK19 over expression were used to modify CDK8/CDK19 activity. MTT assay, propidium iodide staining, wound healing assay, Boyden chamber assay, and adhesion assay were used to investigate cell viability, cell cycle, migration, and adhesion, respectively. Peptide-kinase screen using the PamGene platform was conducted to identify phosphorylated targets. Combining CDK8/CDK19 inhibitors with anti-androgens led to synergistic antiproliferative effects and sensitized androgen-independent cells to bicalutamide. CDK8/CDK19 inhibition resulted in reduced migration and increased collagen I-dependent adhesion. Phosphorylation of multiple peptides linked to cancer progression was identified to be dependent on CDK8/CDK19. In summary, this study substantially supports recent findings on CDK8/ CDK19 in PCa progression. These findings contribute to a better understanding of underlying prooncogenic effects, which is needed to develop CDK8/CDK19 as a therapeutic target in PCa.& nbsp;

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.

Anti-inflammatory treatment of chronic inflammatory diseases often increases susceptibility to infectious diseases such as tuberculosis (TB). Since numerous chronic inflammatory and autoimmune diseases are mediated by interleukin (IL)-6-induced T helper (TH) 17 cells, a TH17-directed anti-inflammatory therapy may be preferable to an IL-12-dependent TH1 inhibition in order to avoid reactivation of latent infections. To assess, however, the risk of inhibition of IL-6-dependent TH17-mediated inflammation, we examined the TH17 immune response and the course of experimental TB in IL-6- and T-cell-specific gp130-deficient mice. Our study revealed that the absence of IL-6 or gp130 on T cells has only a minor effect on the development of antigen-specific TH1 and TH17 cells. Importantly, these gene-deficient mice were as capable as wild type mice to control mycobacterial infection. Together, in contrast to its key function for TH17 development in other inflammatory diseases, IL-6 plays an inferior role for the generation of TH17 immune responses during experimental TB.

Interleukin (IL)-17A-producing T helper (Th)17 cells are increasingly being acknowledged to be associated with protective immunity to Mycobacterium tuberculosis (Mtb). Subunit vaccines potently promote protective immune responses against Mtb infection that correlate with an expansion of IL-23-dependent Th17 cells. Previous studies revealed that after vaccination, IL-23 is required for protection against challenge with Mtb but the underlying IL-23-dependent—and possibly IL-17A-mediated—mechanisms remain elusive. Therefore, we here analyzed the early outcome of Mtb infection in C57BL/6, IL-23p19-deficient ( −/− ), and IL-17A −/− mice after vaccination with the subunit vaccine H1-DDA/TDB to investigate the role of the IL-23-Th17 immune axis for the instruction of vaccine-induced protection. While in IL-23p19 −/− mice the protective effect was reduced, protection after vaccination was maintained in IL-17A −/− animals for the course of infection of 6 weeks, indicating that after vaccination with H1-DDA/TDB early protection against Mtb is—although dependent on IL-23—not mediated by IL-17A. In contrast, IL-17A deficiency appears to have an impact on maintaining long-term protection. In fact, IL-23 instructed the vaccine-induced memory immunity in the lung, in particular the sustained expansion of tumor necrosis factor (TNF) + IL-2 + multifunctional T cells, independently of IL-17A. Altogether, a targeted induction of IL-23 during vaccination against Mtb might improve the magnitude and quality of vaccine-induced memory immune responses. Key messages After subunit Mtb vaccination with H1-DDA/TDB, IL-23 but not IL-17A contributes to vaccine-induced early protection against infection with Mtb. IL-17F does not compensate for IL-17A deficiency in terms of H1-DDA/TDB-induced protection against Mtb infection. IL 23 promotes the H1-DDA/TDB-induced accumulation of effector memory T cells independently of IL 17A. IL-23 arbitrates the induction of H1-specific IFN-γ − TNF + IL-2 + double-positive multifunctional CD4 T cells after subunit Mtb vaccination in an IL-17A-independent manner. Graphical abstract

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.

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.

While persistence in a dormant state is crucial for the life cycle of Mycobacterium tuberculosis , no investigation regarding dormancy survival of different strains across different lineages was performed so far. We analyzed responses to oxygen starvation and recovery in terms of growth, metabolism, and transcription. All different strains belonging to the Euro-American lineage (L4) showed similar survival and resuscitation characteristics. Different clinical isolates from the Beijing (L2), East African-Indian (L3), and Delhi/Central Asian (L1) lineage did not survive oxygen starvation. We show that dormancy survival is lineage-dependent. Recovery from O 2 starvation was only observed in strains belonging to the Euro-American (L4) lineage but not in strains belonging to different lineages (L1, L2, L3). Thus, resuscitation from dormancy after oxygen starvation is not a general feature of all M. tuberculosis strains as thought before. Our findings are of key importance to understand infection dynamics of non-Euro-American vs Euro-American strains and to develop drugs targeting the dormant state.