Image-based quantification of mitochondrial iron uptake via Mitoferrin-2.

Iron is a trace element that is critical for most living organisms and plays a key role in a wide variety of metabolic processes. In the mitochondrion, iron is involved in producing iron-sulfur clusters and synthesis of heme and kept within physiological ranges by concerted activity of multiple molecules. Mitochondrial iron uptake is mediated by the solute carrier transporters Mitoferrin-1 (SLC25A37) and Mitoferrin-2 (SLC25A28). While Mitoferrin-1 is mainly involved in erythropoiesis, the cellular function of the ubiquitously expressed Mitoferrin-2 remains less well defined. Furthermore, Mitoferrin-2 is associated with several human diseases, including cancer, cardiovascular and metabolic diseases, hence representing a potential therapeutic target. Here, we developed a robust approach to quantify mitochondrial iron uptake mediated by Mitoferrin-2 in living cells. We utilize HEK293 cells with inducible expression of Mitoferrin-2 and measure iron-induced quenching of rhodamine B[(1,10-phenanthroline-5-yl)-aminocarbonyl]benzyl ester (RPA) fluorescence and validate this assay for medium-throughput screening. This assay may allow identification and characterization of Mitoferrin-2 modulators and could enable drug discovery for this target.

Ferroportin inhibitor vamifeport ameliorates ineffective erythropoiesis in a mouse model of ß-thalassemia with blood transfusions.

ß-thalassemia is an inherited anemia characterized by ineffective erythropoiesis. Blood transfusions are required for survival in transfusion-dependent ß-thalassemia and are also occasionally needed in patients with non-transfusion-dependent ß-thalassemia. Patients with transfusion-dependent b-thalassemia often have elevated transferrin saturation (TSAT) and non-transferrin-bound iron (NTBI) levels, which can lead to organ iron overload, oxidative stress, and vascular damage. Vamifeport is an oral ferroportin inhibitor that was previously shown to ameliorate anemia, ineffective erythropoiesis, and dysregulated iron homeostasis in the Hbbth3/+ mouse model of ß-thalassemia, under non-transfused conditions. Our study aimed to assess the effects of oral vamifeport on iron-related parameters (including plasma NTBI levels) and ineffective erythropoiesis following blood transfusions in Hbbth3/+ mice. A single dose of vamifeport prevented the transient transfusion-mediated NTBI increase in Hbbth3/+ mice. Compared with vehicle treatment, vamifeport significantly increased hemoglobin levels and red blood cell counts in transfused mice. Vamifeport treatment also significantly improved ineffective erythropoiesis in the spleens of Hbbth3/+ mice, with additive effects observed when treatment was combined with repeated transfusions. Vamifeport corrected leukocyte counts and significantly improved iron-related parameters (serum transferrin, TSAT and erythropoietin levels) versus vehicle treatment in Hbbth3/+ mice, irrespective of transfusion status. In summary, vamifeport prevented transfusion-mediated NTBI formation in Hbbth3/+ mice. When given alone or combined with blood transfusions, vamifeport also ameliorated anemia, ineffective erythropoiesis, and dysregulated iron homeostasis. Administering vamifeport together with repeated blood transfusions additively ameliorated anemia and ineffective erythropoiesis in this mouse model, providing preclinical proof-of-concept for the efficacy of combining vamifeport with blood transfusions in ß-thalassemia.

Structures of ferroportin in complex with its specific inhibitor vamifeport.

A central regulatory mechanism of iron homeostasis in humans involves ferroportin (FPN), the sole cellular iron exporter, and the peptide hormone hepcidin, which inhibits Fe2+ transport and induces internalization and degradation of FPN. Dysregulation of the FPN/hepcidin axis leads to diverse pathological conditions, and consequently, pharmacological compounds that inhibit FPN-mediated iron transport are of high clinical interest. Here, we describe the cryo-electron microscopy structures of human FPN in complex with synthetic nanobodies and vamifeport (VIT-2763), the first clinical-stage oral FPN inhibitor. Vamifeport competes with hepcidin for FPN binding and is currently in clinical development for ß-thalassemia and sickle cell disease. The structures display two distinct conformations of FPN, representing outward-facing and occluded states of the transporter. The vamifeport site is located in the center of the protein, where the overlap with hepcidin interactions underlies the competitive relationship between the two molecules. The introduction of point mutations in the binding pocket of vamifeport reduces its affinity to FPN, emphasizing the relevance of the structural data. Together, our study reveals conformational rearrangements of FPN that are of potential relevance for transport, and it provides initial insight into the pharmacological targeting of this unique iron efflux transporter.

Iron is a modifier of the phenotypes of JAK2-mutant myeloproliferative neoplasms.

JAK 2-V617F mutation causes myeloproliferative neoplasms (MPNs) that can manifest as polycythemia vera (PV), essential thrombocythemia (ET), or primary myelofibrosis. At diagnosis, patients with PV already exhibited iron deficiency, whereas patients with ET had normal iron stores. We examined the influence of iron availability on MPN phenotype in mice expressing JAK2-V617F and in mice expressing JAK2 with an N542-E543del mutation in exon 12 (E12). At baseline, on a control diet, all JAK2-mutant mouse models with a PV-like phenotype displayed iron deficiency, although E12 mice maintained more iron for augmented erythropoiesis than JAK2-V617F mutant mice. In contrast, JAK2-V617F mutant mice with an ET-like phenotype had normal iron stores comparable with that of wild-type (WT) mice. On a low-iron diet, JAK2-mutant mice and WT controls increased platelet production at the expense of erythrocytes. Mice with a PV phenotype responded to parenteral iron injections by decreasing platelet counts and further increasing hemoglobin and hematocrit, whereas no changes were observed in WT controls. Alterations of iron availability primarily affected the premegakaryocyte-erythrocyte progenitors, which constitute the iron-responsive stage of hematopoiesis in JAK2-mutant mice. The orally administered ferroportin inhibitor vamifeport and the minihepcidin PR73 normalized hematocrit and hemoglobin levels in JAK2-V617F and E12 mutant mouse models of PV, suggesting that ferroportin inhibitors and minihepcidins could be used in the treatment for patients with PV.

Functional characterization of SLC39 family members ZIP5 and ZIP10 in overexpressing HEK293 cells reveals selective copper transport activity.

Zinc is the second most prevalent metal element present in living organisms, and control of its concentration is pivotal to physiology. The amount of zinc available to the cell cytoplasm is regulated by the activity of members of the SLC39 family, the ZIP proteins. Selectivity of ZIP transporters has been the focus of earlier studies which provided a biochemical and structural basis for the selectivity for zinc over other metals such as copper, iron, and manganese. However, several previous studies have shown how certain ZIP proteins exhibit higher selectivity for metal elements other than zinc. Sequence similarities suggest an evolutionary basis for the elemental selectivity within the ZIP family. Here, by engineering HEK293 cells to overexpress ZIP proteins, we have studied the selectivity of two phylogenetic clades of ZIP proteins, that is ZIP8/ZIP14 (previously known to be iron and manganese transporters) and ZIP5/ZIP10. By incubating ZIP over-expressing cells in presence of several divalent metals, we found that ZIP5 and ZIP10 are high affinity copper transporters with greater selectivity over other elements, revealing a novel substrate signature for the ZIP5/ZIP10 clade.

The oral ferroportin inhibitor vamifeport improves hemodynamics in a mouse model of sickle cell disease.

Sickle cell disease (SCD) is an inherited hemolytic anemia caused by a single point mutation in the ß-globin gene of hemoglobin that leads to synthesis of sickle hemoglobin (HbS) in red blood cells (RBCs). HbS polymerizes in hypoxic conditions, leading to intravascular hemolysis, release of free hemoglobin and heme, and increased adhesion of blood cells to the endothelial vasculature, which causes painful vaso-occlusion and organ damage. HbS polymerization kinetics are strongly dependent on the intracellular HbS concentration; a relatively small reduction in cellular HbS concentration may prevent HbS polymerization and its sequelae. We hypothesized that iron restriction via blocking ferroportin, the unique iron transporter in mammals, might reduce HbS concentration in RBCs, thereby decreasing hemolysis, improving blood flow, and preventing vaso-occlusive events. Indeed, vamifeport (also known as VIT-2763), a clinical-stage oral ferroportin inhibitor, reduced hemolysis markers in the Townes model of SCD. The RBC indices of vamifeport-treated male and female Townes mice exhibited changes attributable to iron-restricted erythropoiesis: decreased corpuscular hemoglobin concentration mean and mean corpuscular volume, as well as increased hypochromic and microcytic RBC fractions. Furthermore, vamifeport reduced plasma soluble VCAM-1 concentrations, which suggests lowered vascular inflammation. Accordingly, intravital video microscopy of fluorescently labeled blood cells in the microvasculature of Townes mice treated with vamifeport revealed diminished adhesion to the endothelium and improved hemodynamics. These preclinical data provide a strong proof-of-concept for vamifeport in the Townes model of SCD and support further development of this compound as a potential novel therapy in SCD.

Kupffer Cells and Blood Monocytes Orchestrate the Clearance of Iron-Carbohydrate Nanoparticles from Serum.

Intravenous (IV) iron nanoparticle preparations are widely used to treat iron deficiency. The mechanism of mononuclear phagocyte system-mediated clearance of IV iron nanoparticles is unknown. The early uptake and homeostasis of iron after injection of ferric carboxymaltose (FCM) in mice was studied. An increase in serum iron was observed at 2.5 h followed by a return to baseline by 24 h. An increase in circulating monocytes was observed, particularly Ly6Chi and Ly6Clow. FCM was also associated with a time-dependent decrease in liver Kupffer cells (KCs) and increase in liver monocytes. The increase in liver monocytes suggests an influx of iron-rich blood monocytes, while some KCs underwent apoptosis. Adoptive transfer experiments demonstrated that following liver infiltration, blood monocytes differentiated to KCs. KCs were also critical for IV iron uptake and biodegradation. Indeed, anti-Colony Stimulating Factor 1 Receptor (CSF1R)-mediated depletion of KCs resulted in elevated serum iron levels and impaired iron uptake by the liver. Gene expression profiling indicated that C-C chemokine receptor type 5 (CCR5) might be involved in monocyte recruitment to the liver, confirmed by pharmaceutical inhibition of CCR5. Liver KCs play a pivotal role in the clearance and storage of IV iron and KCs appear to be supported by the expanded blood monocyte population.

An Overview of Cell-Based Assay Platforms for the Solute Carrier Family of Transporters.

The solute carrier (SLC) superfamily represents the biggest family of transporters with important roles in health and disease. Despite being attractive and druggable targets, the majority of SLCs remains understudied. One major hurdle in research on SLCs is the lack of tools, such as cell-based assays to investigate their biological role and for drug discovery. Another challenge is the disperse and anecdotal information on assay strategies that are suitable for SLCs. This review provides a comprehensive overview of state-of-the-art cellular assay technologies for SLC research and discusses relevant SLC characteristics enabling the choice of an optimal assay technology. The Innovative Medicines Initiative consortium RESOLUTE intends to accelerate research on SLCs by providing the scientific community with high-quality reagents, assay technologies and data sets, and to ultimately unlock SLCs for drug discovery.

Oral ferroportin inhibitor vamifeport for improving iron homeostasis and erythropoiesis in ß-thalassemia: current evidence and future clinical development.

INTRODUCTION: In ß-thalassemia, imbalanced globin synthesis causes reduced red blood cell survival and ineffective erythropoiesis. Suppressed hepcidin levels increase ferroportin-mediated iron transport in enterocytes, causing increased iron absorption and potentially iron overload. Low hepcidin also stimulates ferroportin-mediated iron release from macrophages, increasing transferrin saturation (TSAT), potentially forming non-transferrin-bound iron, which can be toxic. Modulating the hepcidin-ferroportin axis is an attractive strategy to improve ineffective erythropoiesis and limit the potential tissue damage resulting from iron overload. There are no oral ß-thalassemia treatments that consistently ameliorate anemia and prevent iron overload. AREAS COVERED: The preclinical and clinical development of vamifeport (VIT-2763), a novel ferroportin inhibitor, was reviewed. PubMed, EMBASE and ClinicalTrials.gov were searched using the search term 'VIT-2763'. EXPERT OPINION: Vamifeport is the first oral ferroportin inhibitor in clinical development. In healthy volunteers, vamifeport had comparable safety to placebo, was well tolerated and rapidly decreased iron levels and reduced TSAT, consistent with observations in preclinical models. Data from ongoing/planned Phase II studies are critical to define its potential in ß-thalassemia and other conditions associated with iron overabsorption and/or ineffective erythropoiesis. If vamifeport potentially increases hemoglobin and reduces iron-related parameters, it could be a suitable treatment for non-transfusion-dependent and transfusion-dependent ß-thalassemia.

On Iron Metabolism and Its Regulation.

Iron is a critical metal for several vital biological processes. Most of the body's iron is bound to hemoglobin in erythrocytes. Iron from senescent red blood cells is recycled by macrophages in the spleen, liver and bone marrow. Dietary iron is taken up by the divalent metal transporter 1 (DMT1) in enterocytes and transported to portal blood via ferroportin (FPN), where it is bound to transferrin and taken up by hepatocytes, macrophages and bone marrow cells via transferrin receptor 1 (TfR1). While most of the physiologically active iron is bound hemoglobin, the major storage of most iron occurs in the liver in a ferritin-bound fashion. In response to an increased iron load, hepatocytes secrete the peptide hormone hepcidin, which binds to and induces internalization and degradation of the iron transporter FPN, thus controlling the amount of iron released from the cells into the blood. This review summarizes the key mechanisms and players involved in cellular and systemic iron regulation.

The Oral Ferroportin Inhibitor VIT-2763 Improves Erythropoiesis without Interfering with Iron Chelation Therapy in a Mouse Model of ß-Thalassemia.

In ß-thalassemia, ineffective erythropoiesis leads to anemia and systemic iron overload. The management of iron overload by chelation therapy is a standard of care. However, iron chelation does not improve the ineffective erythropoiesis. We recently showed that the oral ferroportin inhibitor VIT-2763 ameliorates anemia and erythropoiesis in the Hbbth3/+ mouse model of ß-thalassemia. In this study, we investigated whether concurrent use of the iron chelator deferasirox (DFX) and the ferroportin inhibitor VIT-2763 causes any pharmacodynamic interactions in the Hbbth3/+ mouse model of ß-thalassemia. Mice were treated with VIT-2763 or DFX alone or with the combination of both drugs once daily for three weeks. VIT-2763 alone or in combination with DFX improved anemia and erythropoiesis. VIT-2763 alone decreased serum iron and transferrin saturation (TSAT) but was not able to reduce the liver iron concentration. While DFX alone had no effect on TSAT and erythropoiesis, it significantly reduced the liver iron concentration alone and in the presence of VIT-2763. Our results clearly show that VIT-2763 does not interfere with the iron chelation efficacy of DFX. Furthermore, VIT-2763 retains its beneficial effects on improving ineffective erythropoiesis when combined with DFX in the Hbbth3/+ mouse model. In conclusion, co-administration of the oral ferroportin inhibitor VIT-2763 and the iron chelator DFX is feasible and might offer an opportunity to improve both ineffective erythropoiesis and iron overload in ß-thalassemia.

Oral ferroportin inhibitor ameliorates ineffective erythropoiesis in a model of ß-thalassemia.

ß-Thalassemia is a genetic anemia caused by partial or complete loss of ß-globin synthesis, leading to ineffective erythropoiesis and RBCs with a short life span. Currently, there is no efficacious oral medication modifying anemia for patients with ß-thalassemia. The inappropriately low levels of the iron regulatory hormone hepcidin enable excessive iron absorption by ferroportin, the unique cellular iron exporter in mammals, leading to organ iron overload and associated morbidities. Correction of unbalanced iron absorption and recycling by induction of hepcidin synthesis or treatment with hepcidin mimetics ameliorates ß-thalassemia. However, hepcidin modulation or replacement strategies currently in clinical development all require parenteral drug administration. We identified oral ferroportin inhibitors by screening a library of small molecular weight compounds for modulators of ferroportin internalization. Restricting iron availability by VIT-2763, the first clinical stage oral ferroportin inhibitor, ameliorated anemia and the dysregulated iron homeostasis in the Hbbth3/+ mouse model of ß-thalassemia intermedia. VIT-2763 not only improved erythropoiesis but also corrected the proportions of myeloid precursors in spleens of Hbbth3/+ mice. VIT-2763 is currently being developed as an oral drug targeting ferroportin for the treatment of ß-thalassemia.

Adjusted Particle Size Eliminates the Need of Linkage of Antigen and Adjuvants for Appropriated T Cell Responses in Virus-Like Particle-Based Vaccines.

Since the discovery of the first virus-like particle (VLP) derived from hepatitis B virus in 1980 (1), the field has expanded substantially. Besides successful use of VLPs as safe autologous virus-targeting vaccines, the powerful immunogenicity of VLPs has been also harnessed to generate immune response against heterologous and even self-antigens (2-4). Linking adjuvants to VLPs displaying heterologous antigen ensures simultaneous delivery of all vaccine components to the same antigen-presenting cells. As a consequence, antigen-presenting cells, such as dendritic cells, will process and present the antigen displayed on VLPs while receiving costimulatory signals by the VLP-incorporated adjuvant. Similarly, antigen-specific B cells recognizing the antigen linked to the VLP are simultaneously exposed to the adjuvant. Here, we demonstrate in mice that physical association of antigen, carrier (VLPs), and adjuvant is more critical for B than T cell responses. As a model system, we used the E7 protein from human papilloma virus, which spontaneously forms oligomers with molecular weight ranging from 158 kDa to 10 MDa at an average size of 50 nm. E7 oligomers were either chemically linked or simply mixed with VLPs loaded with DNA rich in non-methylated CG motifs (CpGs), a ligand for toll-like receptor 9. E7-specific IgG responses were strongly enhanced if the antigen was linked to the VLPs. In contrast, both CD4+ and CD8+ T cell responses as well as T cell-mediated protection against tumor growth were comparable for linked and mixed antigen formulations. Therefore, our data show that B cell but not T cell responses require antigen-linkage to the carrier and adjuvant for optimal vaccination outcome.

Functional characterization of fluorescent hepcidin.

Hepcidin is a peptide hormone that regulates homeostasis in iron metabolism. It binds to the sole known cellular iron exporter ferroportin (Fpn), triggers its internalization, and thereby modulates the efflux of iron from cells. This functional property has been adopted in this study to assess the bioactivity and potency of a range of novel fluorescent hepcidin analogues. Hepcidin was selectively labeled with 6-carboxyfluorescein (CF) and 6-carboxytetramethylrhodamine (TMR) using Fmoc solid phase peptide chemistry. Internalization of Fpn by hepcidin was assessed by high-content microscopic analysis. Both K18- and M21K-labeled hepcidin with TMR and CF exhibited measurable potency when tested in cultured MDCK and T47D cells expressing human ferroportin. The bioactivity of the labeled hepcidin varies with the type of fluorophore and site of attachment of the fluorophores on the hepcidin molecule.

Memory and effector CD8 T-cell responses after nanoparticle vaccination of melanoma patients.

Induction of cytotoxic CD8 T-cell responses is enhanced by the exclusive presentation of antigen through dendritic cells, and by innate stimuli, such as toll-like receptor ligands. On the basis of these 2 principles, we designed a vaccine against melanoma. Specifically, we linked the melanoma-specific Melan-A/Mart-1 peptide to virus-like nanoparticles loaded with A-type CpG, a ligand for toll-like receptor 9. Melan-A/Mart-1 peptide was cross-presented, as shown in vitro with human dendritic cells and in HLA-A2 transgenic mice. A phase I/II study in stage II-IV melanoma patients showed that the vaccine was well tolerated, and that 14/22 patients generated ex vivo detectable T-cell responses, with in part multifunctional T cells capable to degranulate and produce IFN-γ, TNF-α, and IL-2. No significant influence of the route of immunization (subcutaneous versus intradermal) nor dosing regimen (weekly versus daily clusters) could be observed. It is interesting to note that, relatively large fractions of responding specific T cells exhibited a central memory phenotype, more than what is achieved by other nonlive vaccines. We conclude that vaccination with CpG loaded virus-like nanoparticles is associated with a human CD8 T-cell response with properties of a potential long-term immune protection from the disease.

Mechanisms of allergen-specific desensitization.

BACKGROUND: Allergen-specific desensitization (SIT) is the most effective therapy for allergies. Although allergen-specific antibodies have an important role in the process, mechanisms of IgG-mediated inhibition of allergic reactions are not well defined. OBJECTIVE: We investigated mechanisms by which SIT-induced allergen-specific IgGs inhibit allergic reactions. METHODS: We generated mAbs that recognize 3 nonoverlapping epitopes of the major cat allergen Fel d 1. Each of the mAbs was produced as an IgE and different IgG isotype. RESULTS: IgEs against 2 nonoverlapping epitopes on Fel d 1 are necessary and sufficient to sensitize mast cells for maximal FcepsilonRI signaling and degranulation on exposure to monomeric Fel d 1. IgE antibodies of a third specificity did not further increase mast cell degranulation, indicating that formation of large FcepsilonRI clusters are not required to induce maximal activation of mast cells. A single IgG that was specific for an epitope different from those recognized by the IgEs was a potent inhibitor of Fel d 1-mediated mast cell activation in vitro and in vivo. This inhibition required Fcgamma receptor-IIB. In human beings, IgGs of a single specificity were able to block degranulation of basophils from individuals with cat allergy. The inhibitory potential of these antibodies increased when larger allergen-IgG complexes were formed. CONCLUSIONS: These data reconcile conflicting theories in the literature and might explain the reason IgE levels do not necessarily decrease during therapy, despite clinical efficacy. These findings have important implications for vaccine design.

Cutting edge: limited specialization of dendritic cell subsets for MHC class II-associated presentation of viral particles.

Dendritic cells (DCs) are the most important APC. It was recently reported that there is a dichotomy for Ag presentation by DC subsets; exogenous Ags reach the MHC class I pathway, but not the MHC class II pathway, in CD8(+) DCs, whereas CD8(-) DCs only process Ags for the MHC class II pathway. In this study, we used virus-like particles (VLPs) to show that CD8(+) and CD8(-) DCs efficiently capture and process VLPs for presentation in association with MHC class II in vivo. In contrast, CD8(+) DCs, but not CD8(-) DCs, cross presented VLP-derived peptides. This pattern was changed in an FcgammaR-dependent fashion in the presence of VLP-specific Abs, because under those conditions both DC subsets failed to efficiently cross present. Thus, the presentation of viral particles to CD4(+) T cells is not restricted to distinct DC subsets, whereas the presentation of viral particles to CD8(+) T cells is limited to CD8(+) DCs.

Innate signaling regulates cross-priming at the level of DC licensing and not antigen presentation.

Innate stimuli, such as TLR ligands, are known to greatly facilitate cross-priming. Currently it is unclear whether innate stimuli enhance cross-priming at the level of cross-presentation or at the level of T-cell priming. In this study, we addressed this question by measuring cross-presentation as well as cross-priming by virus-like particles (VLP) displaying peptide p33 derived of lymphocytic choriomeningitis virus. Innate stimuli were varied by either packaging different TLR ligands into virus-like particles or using mice deficient in two key molecules of TLR-signaling, namely the adaptor molecule MyD88 as well as IFN-alpha/beta receptor. While efficient cross-presentation occurred despite strongly reduced activation of DC in the absence of TLR ligand-mediated signals, T-cell priming was abolished. Thus, innate stimuli regulate cross-priming at the level of DC licensing for T-cell activation and not antigen presentation.

Nanoparticles target distinct dendritic cell populations according to their size.

The efficiency of a vaccine largely depends on the appropriate targeting of the innate immune system, mainly through prolonged delivery of antigens and immunomodulatory substances to professional antigen-presenting cells in the lymphoid environment. Particulate antigens, such as virus-like particles (VLP) induce potent immune responses. However, little is known about the relative importance of direct drainage of free antigen to lymph nodes (LN) versus cellular transport and the impact of particle size on the process. Here, we show that nanoparticles traffic to the draining LN in a size-dependent manner. Whereas large particles (500-2000 nm) were mostly associated with dendritic cells (DC) from the injection site, small (20-200 nm) nanoparticles and VLP (30 nm) were also found in LN-resident DC and macrophages, suggesting free drainage of these particles to the LN. In vivo imaging studies in mice conditionally depleted of DC confirmed the capacity of small but not large particles to drain freely to the LN and demonstrated that DC are strictly required for transport of large particles from the injection site to the LN. These data provide evidence that particle size determines the mechanism of trafficking to the LN and show that only small nanoparticles can specifically target LN-resident cells.

Antigen kinetics determines immune reactivity.

A current paradigm in immunology is that the strength of T cell responses is governed by antigen dose, localization, and costimulatory signals. This study investigates the influence of antigen kinetics on CD8 T cell responses in mice. A fixed cumulative antigen dose was administered by different schedules to produce distinct dose-kinetics. Antigenic stimulation increasing exponentially over days was a stronger stimulus for CD8 T cells and antiviral immunity than a single dose or multiple dosing with daily equal doses. The same was observed for dendritic cell vaccination, with regard to T cell and anti-tumor responses, and for T cells stimulated in vitro. In conclusion, stimulation kinetics per se was shown to be a separate parameter of immunogenicity. These findings warrant a revision of current immunization models and have implications for vaccine development and immunotherapy.