The energy sensor AMPK orchestrates metabolic and translational adaptation in expanding T helper cells.

The importance of cellular metabolic adaptation in inducing robust T cell responses is well established. However, the mechanism by which T cells link information regarding nutrient supply to clonal expansion and effector function is still enigmatic. Herein, we report that the metabolic sensor adenosine monophosphate-activated protein kinase (AMPK) is a critical link between cellular energy demand and translational activity and, thus, orchestrates optimal expansion of T cells in vivo. AMPK deficiency did not affect T cell fate decision, activation, or T effector cell generation; however, the magnitude of T cell responses in murine in vivo models of T cell activation was markedly reduced. This impairment was global, as all T helper cell subsets were similarly sensitive to loss of AMPK which resulted in reduced T cell accumulation in peripheral organs and reduced disease severity in pathophysiologically as diverse models as T cell transfer colitis and allergic airway inflammation. T cell receptor repertoire analysis confirmed similar clonotype frequencies in different lymphoid organs, thereby supporting the concept of a quantitative impairment in clonal expansion rather than a skewed qualitative immune response. In line with these findings, in-depth metabolic analysis revealed a decrease in T cell oxidative metabolism, and gene set enrichment analysis indicated a major reduction in ribosomal biogenesis and mRNA translation in AMPK-deficient T cells. We, thus, provide evidence that through its interference with these delicate processes, AMPK orchestrates the quantitative, but not the qualitative, manifestation of primary T cell responses in vivo.

IgG4 induces tolerogenic M2-like macrophages and correlates with disease progression in colon cancer.

IgG4 subclass antibodies are expressed in alternative Th2 environments featuring high IL-10 expression, including several solid tumors such as melanoma. To induce tolerance, allergen immunotherapy mediates antibody class switching from pro-inflammatory IgE to anti-inflammatory IgG4. We previously reported that IgG4 drives allergic M2 macrophages toward tolerogenic states. Here we assessed the roles of IgG4 and macrophage activation in colorectal cancer (CRC). In this observer-blinded, case-control study, we analyzed total circulating serum IgE, IgG1 and IgG4 levels in CRC (n = 38) patients with (n = 13, TxNxM1) or without (n = 25, TxNxM0) metastasis, and in healthy donors (n = 21). Primary cultures of circulating monocyte-derived macrophages from healthy controls and CRC patients were further evaluated in their responses to stimulation with IgG1 or IgG4. We found higher absolute serum levels of IgG4 in patients with CRC. IgG4 enabled polarization of macrophages derived from CRC patients and healthy controls into alternatively-activated tolerogenic M2b phenotypes. IgG4-stimulated M2 macrophages were characterized by lower surface CD206, CD163, CD14, and CD11b expression and higher CCL-1, IL-10, and IL-6 production. IgG4 was less potent that IgG1 in triggering antibody-dependent cell-mediated phagocytosis (ADCP) of cancer cells. Further, higher z-normalized IgG4/-IgE sera level ratios correlated with the presence of metastasis (p = .0247 and p = .0009, respectively) in CRC patients. High IgG4 in CRC synergizes with macrophages in shaping an immunosuppressive microenvironment and impairs anti-cancer effector cell functions. The shift of serum IgG4/IgE ratios toward enhanced tolerance induction in metastatic disease indicates a role for high IgG4 in disease progression and poor prognostic outcome.

Cesarean section and risk of allergies in Ecuadorian children: A cross-sectional study.

BACKGROUND: Studies have shown an association between cesarean section (CS) and increased prevalence of childhood allergic diseases. While these observations have been consistent in industrialized countries, evidence from developing countries is limited. OBJECTIVE: To assess the association between the mode of delivery and allergic diseases in children aged 3-12 years in Quito, Ecuador. METHODS: In this cross-sectional study, parents were surveyed using an anonymous, standardized questionnaire from the International Study of Asthma and Allergies in Childhood project to assess the presence of asthma, allergic rhinitis, atopic dermatitis, and food allergies in their children. The children's age, sex, birthplace, delivery mode (CS/vaginal), socioeconomic status, and ethnicity were recorded. Other parameters included gestational age, breastfeeding, smoking status during pregnancy, and parental allergic diseases. RESULTS: After adjusting for confounding factors, children delivered via CS were found to have a higher risk of wheezing (odds ratio [OR] = 4.12, 95% confidence interval [CI]: 1.43-11.89), physician-diagnosed asthma (OR = 24.06; 95% CI: 1.98-292.3), and pimples, or eczema with the itching for 6 months (OR = 2.65; 95% CI: 1.06-6.61) than children delivered vaginally. No association was found between the delivery mode and rhinitis or food allergies. After stratifying by socioeconomic status, CS was only associated with allergic disorders in children of medium/high socioeconomic backgrounds. CONCLUSIONS: As seen in industrialized settings, children born by CS in nonindustrialized countries have an increased risk of developing allergic disorders including asthma and dermatitis, compared to those delivered vaginally.

Transferrin receptor 1 is a cellular receptor for human heme-albumin.

Iron is essential for living cells. Uptake of iron-loaded transferrin by the transferrin receptor 1 (CD71, TFR) is a major but not sufficient mechanism and an alternative iron-loaded ligand for CD71 has been assumed. Here, we demonstrate that CD71 utilizes heme-albumin as cargo to transport iron into human cells. Binding and endocytosis of heme-albumin via CD71 was sufficient to promote proliferation of various cell types in the absence of transferrin. Growth and differentiation of cells induced by heme-albumin was dependent on heme-oxygenase 1 (HO-1) function and was accompanied with an increase of the intracellular labile iron pool (LIP). Import of heme-albumin via CD71 was further found to contribute to the efficacy of albumin-based drugs such as the chemotherapeutic Abraxane. Thus, heme-albumin/CD71 interaction is a novel route to transport nutrients or drugs into cells and adds to the emerging function of CD71 as a scavenger receptor.

Iron Deprivation in Human T Cells Induces Nonproliferating Accessory Helper Cells.

Iron uptake via the transferrin receptor (CD71) is a pivotal mechanism for T cell proliferation. Yet, it is incompletely understood if targeting of CD71 also affects the differentiation and functional polarization of primary human T cells. In this study, we demonstrate that inhibition of iron ingestion with blocking mAbs against CD71 induces nonproliferating T cells, which release high amounts of IL-2. Targeting of CD71 with blocking or nonblocking mAbs did not alter major signaling pathways and the activation of the transcription factors NF-κB, NFAT, or AP-1 as analyzed in Jurkat T cells. Growth arrest in iron-deficient (Fe-def) T cells was prevented upon addition of exogenous iron in the form of ferric ammonium citrate but was not reversible by exogenous IL-2. Surprisingly, protein synthesis was found to be intact in Fe-def T cells as demonstrated by comparable levels of CD69 upregulation and cytokine production with iron-sufficient T cells upon stimulation with CD3 plus CD28 mAbs. Indeed, high amounts of IL-2 were detectable in the supernatant of Fe-def T cells, which was accompanied with a reduced cell surface expression of IL-2R. When we used such Fe-def T cells in allogeneic MLRs, we observed that these cells acquired an accessory cell function and stimulated the proliferation of bystander T cells by providing IL-2. Thus, the results of our study demonstrate that iron deprivation causes nonproliferating, altruistic T cells that can help and stimulate other immune cells by providing cytokines such as IL-2.

TIM-3 and CEACAM1 do not interact in cis and in trans.

TIM-3 has been considered as a target in cancer immunotherapy. In T cells, inhibitory as well as activating functions have been ascribed to this molecule. Its role may therefore depend on the state of T cells and on the presence of interaction partners capable to perform functional pairing. Carcinoembryonic antigen-related cell adhesion molecule (CEACAM1) has been proposed to bind TIM-3 and to regulate its function. Using a T cell reporter platform we confirmed CEACAM1-mediated inhibition, but CEACAM1 did not functionally engage TIM-3. TIM-3 and CEACAM1 coexpression was limited to a small subset of activated T cells. Moreover, results obtained in extensive binding studies were not in support of an interaction between TIM-3 and CEACAM1. Cytoplasmic sequences derived from TIM-3 induced inhibitory signaling in our human T cell reporter system. Our results indicate that TIM-3 functions are independent of CEACAM1 and that this receptor has the capability to promote inhibitory signaling pathways in human T cells.

Low Systemic Levels of Chemokine C-C Motif Ligand 3 (CCL3) are Associated with a High Risk of Venous Thromboembolism in Patients with Glioma.

A tight interplay between inflammation and hemostasis has been described as a potential driver for developing venous thromboembolism (VTE). Here, we investigated the association of systemic cytokine levels and risk of VTE in patients with glioma. This analysis was conducted within the prospective, observational Vienna Cancer and Thrombosis Study. Patients with glioma were included at time of diagnosis or progression and were observed for a maximum of two years. Primary endpoint was objectively confirmed VTE. At study entry, a single blood draw was performed. A panel of nine cytokines was measured in serum samples with the xMAP technology developed by Luminex. Results: Overall, 76 glioma patients were included in this analysis, and 10 (13.2%) of them developed VTE during the follow-up. Chemokine C-C motif ligand 3 (CCL3) levels were inversely associated with risk of VTE (hazard ratio [HR] per double increase, 95% confidence interval [CI]: 0.385, 95% CI: 0.161-0.925, p = 0.033), while there was no association between the risk of VTE and serum levels of interleukin (IL)-1ß, IL-4, IL-6, IL-8, IL-10, IL-11, tumor necrosis factor (TNF)-α and vascular endothelial growth factor (VEGF), respectively. In conclusion, low serum levels of CCL3 were associated with an increased risk of VTE. CCL3 might serve as a potential biomarker to predict VTE risk in patients with glioma.

Neuropilin-1 Acts as a Receptor for Complement Split Products.

Complement split products (CSPs), such as the fragments C4d and C3d, which are generated as a consequence of complement regulatory processes, are established markers for disease activity in autoimmunity or antibody-mediated graft rejection. Since immunoglobulin-like transcript 4 (ILT4) was previously shown to interact with soluble CSPs, but not with CSPs covalently-bound to target surfaces following classical complement activation, the present study aimed to identify novel cellular receptors interacting with covalently-deposited CSPs. By applying an unbiased screening approach using a cDNA mammalian expression library generated from human monocyte-derived dendritic cells and probed with recombinant human C4d, we identified neuropilin-1 (NRP1) as a novel receptor for C4d, C3d, and iC3b. NRP1, a highly conserved type 1 transmembrane protein, plays important roles in the development of the nervous and cardiovascular system as well as in tumorigenesis through interaction with its established binding partners, such as vascular endothelial growth factor (VEGF) and semaphorin 3A (Sema3A). NRP1 is also expressed on immune cells and serves as a marker for murine Tregs. Although NRP1 contains domains homologous to ones found in some complement proteins, it has not been linked to the complement system. We demonstrate that binding of C4d to NRP1 expressing cells was dose-dependent and saturable, and had a KD value of 0.71 µM. Importantly, and in contrast to ILT4, NRP1 interacted with CSPs that were covalently bound to target surfaces in the course of complement activation, therefore representing a classical complement receptor. The binding site of CSPs was mapped to the b1 domain of the coagulation factor V/VIII homology domain of NRP1. Taken together, our results demonstrate a novel role for NRP1 as a receptor for CSPs deposited on surfaces during complement activation. Further work is required to elucidate the functional consequences of the NRP1-CSP interactions in immunity.

Therapeutic PD-L1 antibodies are more effective than PD-1 antibodies in blocking PD-1/PD-L1 signaling.

Inhibitors of PD-1 signaling have revolutionized cancer therapy. PD-1 and PD-L1 antibodies have been approved for the treatment of cancer. To date, therapeutic PD-1 inhibitors have not been compared in a functional assay. We used an efficient T cell reporter platform to evaluate the efficacy of five clinically used PD-1 inhibitors to block PD-1 signaling. The half maximal effective concentrations (EC50) for nivolumab and pembrolizumab were 76.17 ng/ml (95% CI 64.95-89.34 ng/ml) and 39.90 ng/ml (34.01-46.80 ng/ml), respectively. The EC50 values of the PD-L1 inhibitors were 6.46 ng/ml (5.48-7.61 ng/ml), 6.15 ng/ml (5.24-7.21 ng/ml) and 7.64 ng/ml (6.52-8.96 ng/ml) for atezolizumab, avelumab, and durvalumab, respectively. In conclusion, a functional assay evaluating antibodies targeting PD-1 inhibition in vitro revealed that pembrolizumab is a slightly more effective PD-1 blocker than nivolumab, and that PD-L1 antibodies are superior to PD-1 antibodies in reverting PD-1 signaling.

CTLA-4 antibody ipilimumab negatively affects CD4+ T-cell responses in vitro.

Immune checkpoint inhibitors targeting coinhibitory pathways in T cells possess efficacy in combating cancer. In addition to PD-1/PD-L1 and CTLA-4 antibodies which are already established in tumor immunotherapy, immune checkpoints such as LAG-3 or BTLA are emerging, which may have the potential to enhance T-cell responses alone or in combination with PD-1 blockers. CD4+ T cells play a central role in the immune system and contribute to productive immune responses in multiple ways. The effects of immune checkpoint inhibitors on this cell subset may thus critically influence therapeutic outcomes. Here, we have used in vitro responses to tetanus toxoid (TT) as a model system to study the effects of immune checkpoint inhibitors on CD4+ T-cell responses. CFSE-labeled PBMCs of 65 donors were stimulated with TT in the presence of blocking antibodies to PD-L1, CTLA-4, LAG-3, or BTLA for 7 days. We found that the PD-L1 antibody greatly enhanced cytokine production and antigen-specific CD4+ T-cell proliferation, whereas blocking antibodies to BTLA or LAG-3 did not augment responses to TT. Surprisingly, the presence of the therapeutic CTLA-4 antibody ipilimumab resulted in a significant reduction of CD4+ T-cell proliferation and cytokine production. Stimulation experiments with an IgG4 variant of ipilimumab indicated that the inhibitory effect of ipilimumab was dependent on its IgG1 isotype. Our results indicate that the therapeutic CTLA-4 antibody ipilimumab can impair CD4+ effector T-cell responses and that this activity is mediated by its Fc part and CD16-expressing cells.

T-cell-derived cytokines enhance the antigen-presenting capacity of human neutrophils.

Activated allergen-specific Th2 and Th1 cells release cytokines that transform neutrophils into functional APCs characterized by the expression of HLA-DR and CD58 as well as enhanced survival and antigen uptake, irrespectively of the presence of IL-10, which reduces allergen uptake by neutrophils.

Hijacking the Supplies: Metabolism as a Novel Facet of Virus-Host Interaction.

Viral replication is a process that involves an extremely high turnover of cellular molecules. Since viruses depend on the host cell to obtain the macromolecules needed for their proper replication, they have evolved numerous strategies to shape cellular metabolism and the biosynthesis machinery of the host according to their specific needs. Technologies for the rigorous analysis of metabolic alterations in cells have recently become widely available and have greatly expanded our knowledge of these crucial host-pathogen interactions. We have learned that most viruses enhance specific anabolic pathways and are highly dependent on these alterations. Since uninfected cells are far more plastic in their metabolism, targeting of the virus-induced metabolic alterations is a promising strategy for specific antiviral therapy and has gained great interest recently. In this review, we summarize the current advances in our understanding of metabolic adaptations during viral infections, with a particular focus on the utilization of this information for therapeutic application.

A novel role for neutrophils in IgE-mediated allergy: Evidence for antigen presentation in late-phase reactions.

BACKGROUND: Neutrophils and allergen-specific T cells accumulate in patients with allergic late-phase reactions (LPRs). Their presence is associated with severe inflammation. Cytokines, such as GM-CSF, IFN-γ, and IL-3, which are typically found in patients with allergic LPRs, have been proposed to convert neutrophils into antigen-presenting cells (APCs). OBJECTIVE: We sought to assess the antigen-processing and antigen-presenting capacities of neutrophils from allergic patients. METHODS: Neutrophils were isolated from peripheral blood of donors with birch pollen allergy and stimulated with GM-CSF, IFN-γ, and IL-3. The viability and expression of HLA-DR, CD80, and CD86 were assessed by using flow cytometry. HLA-DM expression was analyzed by means of immunoblotting. Allergen uptake was studied after fluorescence labeling of the major birch pollen allergen Bet v 1. Bet v 1 was digested with neutrophilic endolysosomal extracts, and the resulting fragments were sequenced by using mass spectrometry. Neutrophils were used as APCs in coculture experiments with autologous HLA-DR-restricted and Bet v 1-specific T-cell clones reactive with epitopes in different regions of the allergen. In all experiments monocytes were used for comparison. Fluids from suction blisters formed on top of LPRs induced by using intradermal allergen injection were assessed for HLA-DR+ neutrophils by using flow cytometry. RESULTS: The cytokines significantly enhanced the survival, allergen uptake, and expression of HLA-DM and HLA-DR on neutrophils. Neutrophils rapidly degraded Bet v 1 into fragments containing all relevant T-cell epitopes. Cytokine-activated, allergen-pulsed neutrophils induced proliferative and cytokine responses of Bet v 1-specific T cells irrespective of epitope specificity, confirming that they fully processed and presented the allergen. HLA-DR+ neutrophils were detected in patients with cutaneous allergic LPRs. CONCLUSION: Neutrophils can serve as APCs for local allergen-specific effector T cells in patients with allergic LPRs.

PD-1 has a unique capacity to inhibit allergen-specific human CD4+ T cell responses.

T lymphocytes have a crucial role in initiating and promoting type I allergies. Their responses are tightly regulated by numerous activating and inhibitory signals provided by APCs. Here we have addressed the role of the major coinhibitory receptors PD-1, CTLA-4, BTLA and LAG-3 in allergen-specific CD4+ T cell responses. PBMCs of healthy individuals and 41 patients allergic to house dust mites, birch, grass or mugwort pollen were stimulated with allergenic extracts and expression of coinhibitory receptors on responding CD4+ T cells was assessed. Blocking antibodies to PD-1, CTLA-4, BTLA and LAG-3 were used to evaluate the role of coinhibitory pathways. Allergen-specific CD4+ T cells showed strong upregulation of PD-1, LAG-3 and CTLA-4 upon stimulation, whereas BTLA was downregulated. Blockade of PD-1 strongly enhanced proliferation and cytokine production (IL-10; TH1 cytokines IFN-γ, TNF-α; TH2 cytokines IL-5, IL-13) of allergen-specific CD4+ T cells derived from allergic as well as non-allergic individuals. BTLA blockade enhanced proliferation but not cytokine production in response to house dust mite extract. Blocking LAG-3 was ineffective and surprisingly, we observed reduced proliferation and cytokine production in presence of a CTLA-4 antibody. Our results point to a unique potency of PD-1 pathways to dampen allergen-specific human T cells.

Local and systemic levels of cytokines and danger signals in endometriosis-affected women.

Endometriosis is a prevalent gynaecological disorder with a still unclear pathogenesis. So far inflammatory mechanisms are associated with disease progression and critical reviews have discussed the so-called 'danger theory' related to endometriosis. Hence, we performed immunoassays to evaluate whether local inflammation is linked to the severity of the disease. In addition, we investigated the role of recently described cytokines IL-33, IL-32α and the 'alarmin' high mobility group box 1 (HMGB1). We confirmed a dysfunctional immune response in the local environment of women suffering from endometriosis. However, we found no direct evidence for a significant up-regulation of danger signals in endometriosis, irrespective of the severity of the disease.

Rhinovirus induces an anabolic reprogramming in host cell metabolism essential for viral replication.

Rhinoviruses (RVs) are responsible for the majority of upper airway infections; despite their high prevalence and the resulting economic burden, effective treatment is lacking. We report here that RV induces metabolic alterations in host cells, which offer an efficient target for antiviral intervention. We show that RV-infected cells rapidly up-regulate glucose uptake in a PI3K-dependent manner. In parallel, infected cells enhance the expression of the PI3K-regulated glucose transporter GLUT1. In-depth metabolomic analysis of RV-infected cells revealed a critical role of glucose mobilization from extracellular and intracellular pools via glycogenolysis for viral replication. Infection resulted in a highly anabolic state, including enhanced nucleotide synthesis and lipogenesis. Consistently, we observed that glucose deprivation from medium and via glycolysis inhibition by 2-deoxyglucose (2-DG) potently impairs viral replication. Metabolomic analysis showed that 2-DG specifically reverts the RV-induced anabolic reprogramming. In addition, treatment with 2-DG inhibited RV infection and inflammation in a murine model. Thus, we demonstrate that the specific metabolic fingerprint of RV infection can be used to identify new targets for therapeutic intervention.

Extracellular Purine Metabolism Is the Switchboard of Immunosuppressive Macrophages and a Novel Target to Treat Diseases With Macrophage Imbalances.

If misregulated, macrophage (Mϕ)-T cell interactions can drive chronic inflammation thereby causing diseases, such as rheumatoid arthritis (RA). We report that in a proinflammatory environment, granulocyte-Mϕ (GM-CSF)- and Mϕ colony-stimulating factor (M-CSF)-dependent Mϕs have dichotomous effects on T cell activity. While GM-CSF-dependent Mϕs show a highly stimulatory activity typical for M1 Mϕs, M-CSF-dependent Mϕs, marked by folate receptor ß (FRß), adopt an immunosuppressive M2 phenotype. We find the latter to be caused by the purinergic pathway that directs release of extracellular ATP and its conversion to immunosuppressive adenosine by co-expressed CD39 and CD73. Since we observed a misbalance between immunosuppressive and immunostimulatory Mϕs in human and murine arthritic joints, we devised a new strategy for RA treatment based on targeted delivery of a novel methotrexate (MTX) formulation to the immunosuppressive FRß+CD39+CD73+ Mϕs, which boosts adenosine production and curtails the dominance of proinflammatory Mϕs. In contrast to untargeted MTX, this approach leads to potent alleviation of inflammation in the murine arthritis model. In conclusion, we define the Mϕ extracellular purine metabolism as a novel checkpoint in Mϕ cell fate decision-making and an attractive target to control pathological Mϕs in immune-mediated diseases.

Allergic patients with and without allergen-specific immunotherapy mount protective immune responses to tick-borne encephalitis vaccination in absence of enhanced side effects or propagation of their Th2 bias.

BACKGROUND: Allergic diseases are caused by Th2-driven immune responses and their treatment with specific immunotherapy (SIT) leads to immunomodulation via IL10, TGF-ß and Th1/Tr1 shift. This phase IV, open-label clinical trial investigated whether allergies and SIT treatment influenced immune responses to routine vaccination. METHODS: We studied three groups: 49 allergic patients (allergic group), 21 allergic patients receiving maintenance doses of SIT (SIT group), and 49 non-allergic controls. All subjects received tick-borne encephalitis (TBE) booster vaccines and humoral and cellular immune responses were evaluated after one week, four weeks and six months. RESULTS: The levels and kinetics of neutralizing TBE-specific antibodies, reflecting protection against TBE, were not significantly different in the three groups. The allergic group showed Th2 polarization pre-booster as indicated by increased TBE-specific IgG1 and elevated mitogen-induced IL5 production. Alum-adjuvanted TBE vaccine led to Th2 biased immune responses in the controls, but to no further enhancement of Th2 polarization in the allergic and SIT group. Furthermore, in the SIT group cellular parameters reflected the induction of immunomodulation due to increased Tregs, elevated baseline IL10 and lack of TBE-specific IL5. Importantly, these cellular regulatory responses did not limit the ability to mount sufficient TBE-specific antibodies after the booster. All groups tolerated the vaccine well with no exacerbation of allergic symptoms. CONCLUSION: TBE booster vaccinations were immunogenic and safe in both the allergic and SIT group and contributed to balanced immune responses. Our data indicate that all allergic patients, even when undergoing SIT, should be vaccinated without hesitation and at regular intervals according to standard recommendations. ClinicalTrials.gov (NCT02511535).

Fusion proteins of flagellin and the major birch pollen allergen Bet v 1 show enhanced immunogenicity, reduced allergenicity, and intrinsic adjuvanticity.

BACKGROUND: Recombinant fusion proteins of flagellin and antigens have been demonstrated to induce strong innate and adaptive immune responses. Such fusion proteins can enhance the efficacy of allergen-specific immunotherapy. OBJECTIVE: We sought to characterize different fusion proteins of flagellin and the major birch pollen allergen Bet v 1 for suitability as allergy vaccines. METHODS: A truncated version of flagellin (NtCFlg) was genetically fused to the N- or C-terminus of Bet v 1. Toll-like receptor (TLR) 5 binding was assessed with HEK293 cells expressing TLR5. Upregulation of CD40, CD80, CD83, and CD86 on monocyte-derived dendritic cells from allergic patients was analyzed by using flow cytometry. The T cell-stimulatory capacity of the fusion proteins was assessed with naive and Bet v 1-specific T cells. IgE binding was tested in inhibition ELISAs and basophil activation tests. Mice were immunized with the fusion proteins in the absence and presence of aluminum hydroxide. Cellular and antibody responses were monitored. Murine antibodies were tested for blocking capacity in basophil activation tests. RESULTS: Both fusion proteins matured monocyte-derived dendritic cells through TLR5. Compared with Bet v 1, the fusion proteins showed stronger T cell-stimulatory and reduced IgE-binding capacity and induced murine Bet v 1-specific antibodies in the absence of aluminum hydroxide. However, only antibodies induced by means of immunization with NtCFlg fused to the C-terminus of Bet v 1 inhibited binding of patients' IgE antibodies to Bet v 1. CONCLUSION: Bet v 1-flagellin fusion proteins show enhanced immunogenicity, reduced allergenicity, and intrinsic adjuvanticity and thus represent promising vaccines for birch pollen allergen-specific immunotherapy. However, the sequential order of allergen and adjuvant within a fusion protein determines its immunologic characteristics.