RESUMEN
Aging is associated with dysregulated immune functions. Here, we investigated the impact of age on neutrophil diapedesis. Using confocal intravital microscopy, we found that in aged mice, neutrophils adhered to vascular endothelium in inflamed tissues but exhibited a high frequency of reverse transendothelial migration (rTEM). This retrograde breaching of the endothelium by neutrophils was governed by enhanced production of the chemokine CXCL1 from mast cells that localized at endothelial cell (EC) junctions. Increased EC expression of the atypical chemokine receptor 1 (ACKR1) supported this pro-inflammatory milieu in aged venules. Accumulation of CXCL1 caused desensitization of the chemokine receptor CXCR2 on neutrophils and loss of neutrophil directional motility within EC junctions. Fluorescent tracking revealed that in aged mice, neutrophils undergoing rTEM re-entered the circulation and disseminated to the lungs where they caused vascular leakage. Thus, neutrophils stemming from a local inflammatory site contribute to remote organ damage, with implication to the dysregulated systemic inflammation associated with aging.
Asunto(s)
Envejecimiento/inmunología , Transporte Biológico/inmunología , Inflamación/inmunología , Neutrófilos/inmunología , Animales , Quimiocina CXCL1/inmunología , Células Endoteliales/inmunología , Endotelio Vascular/inmunología , Femenino , Uniones Intercelulares/inmunología , Pulmón/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de Interleucina-8B/inmunología , Vénulas/inmunologíaRESUMEN
Neutrophils require directional cues to navigate through the complex structure of venular walls and into inflamed tissues. Here we applied confocal intravital microscopy to analyze neutrophil emigration in cytokine-stimulated mouse cremaster muscles. We identified differential and non-redundant roles for the chemokines CXCL1 and CXCL2, governed by their distinct cellular sources. CXCL1 was produced mainly by TNF-stimulated endothelial cells (ECs) and pericytes and supported luminal and sub-EC neutrophil crawling. Conversely, neutrophils were the main producers of CXCL2, and this chemokine was critical for correct breaching of endothelial junctions. This pro-migratory activity of CXCL2 depended on the atypical chemokine receptor 1 (ACKR1), which is enriched within endothelial junctions. Transmigrating neutrophils promoted a self-guided migration response through EC junctions, creating a junctional chemokine "depot" in the form of ACKR1-presented CXCL2 that enabled efficient unidirectional luminal-to-abluminal migration. Thus, CXCL1 and CXCL2 act in a sequential manner to guide neutrophils through venular walls as governed by their distinct cellular sources.
Asunto(s)
Quimiocina CXCL1 , Quimiocina CXCL2 , Sistema del Grupo Sanguíneo Duffy , Neutrófilos , Receptores de Superficie Celular , Migración Transendotelial y Transepitelial , Animales , Músculos Abdominales/efectos de los fármacos , Músculos Abdominales/inmunología , Músculos Abdominales/metabolismo , Quimiocina CXCL1/genética , Quimiocina CXCL1/inmunología , Quimiocina CXCL1/metabolismo , Quimiocina CXCL2/genética , Quimiocina CXCL2/inmunología , Quimiocina CXCL2/metabolismo , Sistema del Grupo Sanguíneo Duffy/genética , Sistema del Grupo Sanguíneo Duffy/inmunología , Sistema del Grupo Sanguíneo Duffy/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Uniones Intercelulares/efectos de los fármacos , Uniones Intercelulares/inmunología , Uniones Intercelulares/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neutrófilos/citología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Receptores de Superficie Celular/metabolismo , Migración Transendotelial y Transepitelial/efectos de los fármacos , Migración Transendotelial y Transepitelial/genética , Migración Transendotelial y Transepitelial/inmunología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Chronic lymphocytic leukemia (CLL) outgrowth depends on signals from the microenvironment. We have previously found that in vitro reconstitution of this microenvironment induces specific variant isoforms of the adhesion molecule CD44, which confer human CLL with high affinity to hyaluronan (HA). Here, we determined the in vivo contribution of standard CD44 and its variants to leukemic B-cell homing and proliferation in Tcl1 transgenic mice with a B-cell-specific CD44 deficiency. In these mice, leukemia onset was delayed and leukemic infiltration of spleen, liver, and lungs, but not of bone marrow, was decreased. Competitive transplantation revealed that CLL homing to spleen and bone marrow required functional CD44. Notably, enrichment of CD44v6 variants particularly in spleen enhanced CLL engraftment and proliferation, along with increased HA binding. We recapitulated CD44v6 induction in the human disease and revealed the involvement of MAPK and NF-κB signaling upon CD40 ligand and B-cell receptor stimulation by in vitro inhibition experiments and chromatin immunoprecipitation assays. The investigation of downstream signaling after CD44v6-HA engagement uncovered the activation of extracellular signal-regulated kinase and p65. Consequently, anti-CD44v6 treatment reduced leukemic cell proliferation in vitro in human and mouse, confirming the general nature of the findings. In summary, we propose a CD44-NF-κB-CD44v6 circuit in CLL, allowing tumor cells to gain HA binding capacity and supporting their proliferation.
Asunto(s)
Proliferación Celular , Receptores de Hialuranos/metabolismo , Leucemia Linfocítica Crónica de Células B/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas de Neoplasias/metabolismo , Microambiente Tumoral , Animales , Humanos , Receptores de Hialuranos/genética , Ácido Hialurónico/genética , Ácido Hialurónico/metabolismo , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Ratones , Ratones Transgénicos , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas de Neoplasias/genética , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/metabolismo , Bazo/metabolismo , Bazo/patologíaRESUMEN
Human neutrophil elastase is an important regulator of the immune response and plays a role in host defense mechanisms and further physiological processes. The uncontrolled activity of this serine protease may cause severe tissue alterations and impair inflammatory states. The design of an activity-based probe for human neutrophil elastase reported herein relies on a sulfonyloxyphthalimide moiety as a new type of warhead that is linker-connected to a coumarin fluorophore. The inhibitory potency of the activity-based probe was assessed against several serine and cysteine proteases, and the selectivity for human neutrophil elastase (Ki = 6.85 nM) was determined. The adequate fluorescent tag of the probe allowed for the in-gel fluorescence detection of human neutrophil elastase in the low nanomolar range. The coumarin moiety and the anthranilic acid function of the probe, produced in the course of a Lossen rearrangement, were part of two different Förster resonance energy transfers.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/análisis , Elastasa de Leucocito/análisis , Animales , Bovinos , Proteasas de Cisteína/metabolismo , Glicina/análogos & derivados , Glicina/farmacología , Células HEK293 , Humanos , Elastasa de Leucocito/antagonistas & inhibidores , Activación Neutrófila , Neutrófilos/enzimología , Serina Proteasas/metabolismo , Sulfonamidas/farmacología , PorcinosRESUMEN
BACKGROUND: Granulocyte-colony-stimulating factor (G-CSF) is routinely used for mobilization of hematopoietic stem and progenitor cells preceding autologous transplantation after high-dose chemotherapy in hematologic malignancies. However, due to high mobilization failure rates, alternative mobilization strategies are required. STUDY DESIGN AND METHODS: Patients who poorly mobilized CD34+ hematopoietic cells (HCs) with G-CSF additionally received the CXCR4 antagonist plerixafor. The phenotype of CD34+ HCs collected after this plerixafor-induced "rescue" mobilization, in regard to adhesion molecule and CD133, CD34, and CD38 expression in comparison to CD34+ HCs collected after traditional G-CSF administration in good mobilizers, was analyzed flow cytometrically. To confirm previous studies in our patient cohort, the efficiency of mobilization and subsequent engraftment after this "on-demand" plerixafor mobilization were analyzed. RESULTS: Pronounced mobilization occurred after plerixafor administration in poor mobilizers, resulting in similar CD34+ cell yields as obtained by G-CSF in good mobilizers, whereby plerixafor increased the content of primitive CD133+/CD34+/CD38- cells. The surface expression profiles of the marrow homing and retention receptors CXCR4, VLA-4, LFA-1, and CD44 on mobilized CD34+ cells and hematopoietic recovery after transplantation were similar in patients receiving G-CSF plus plerixafor or G-CSF. Unexpectedly, the expression levels of respective adhesion receptors were not related to mobilization efficiency or engraftment. CONCLUSION: The results show that CD34+ HCs collected by plerixafor-induced rescue mobilization are qualitatively equivalent to CD34+ HCs collected after traditional G-CSF mobilization in good mobilizers, in regard to their adhesive phenotype and engraftment potential. Thereby, plerixafor facilitates the treatment of poor mobilizers with autologous HC transplantation after high-dose chemotherapy.
Asunto(s)
Antígenos CD34/metabolismo , Factor Estimulante de Colonias de Granulocitos/farmacología , Compuestos Heterocíclicos/farmacología , Receptores CXCR4/metabolismo , Adolescente , Adulto , Anciano , Bencilaminas , Ciclamas , Femenino , Movilización de Célula Madre Hematopoyética , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Receptores CXCR4/antagonistas & inhibidores , Adulto JovenRESUMEN
Neutrophil diapedesis is an immediate step following infections and injury and is driven by complex interactions between leukocytes and various components of the blood vessel wall. Here, we show that perivascular mast cells (MC) are key regulators of neutrophil behaviour within the sub-endothelial space of inflamed venules. Using confocal intravital microscopy, we observe directed abluminal neutrophil motility along pericyte processes towards perivascular MCs, a response that created neutrophil extravasation hotspots. Conversely, MC-deficiency and pharmacological or genetic blockade of IL-17A leads to impaired neutrophil sub-endothelial migration and breaching of the pericyte layer. Mechanistically, identifying MCs as a significant cellular source of IL-17A, we establish that MC-derived IL-17A regulates the enrichment of key effector molecules ICAM-1 and CXCL1 in nearby pericytes. Collectively, we identify a novel MC-IL-17A-pericyte axis as modulator of the final steps of neutrophil diapedesis, with potential translational implications for inflammatory disorders driven by increased neutrophil diapedesis.
Asunto(s)
Neutrófilos , Migración Transendotelial y Transepitelial , Neutrófilos/fisiología , Pericitos , Interleucina-17 , MastocitosRESUMEN
Increased microvascular permeability to plasma proteins and neutrophil emigration are hallmarks of innate immunity and key features of numerous inflammatory disorders. Although neutrophils can promote microvascular leakage, the impact of vascular permeability on neutrophil trafficking is unknown. Here, through the application of confocal intravital microscopy, we report that vascular permeability-enhancing stimuli caused a significant frequency of neutrophil reverse transendothelial cell migration (rTEM). Furthermore, mice with a selective defect in microvascular permeability enhancement (VEC-Y685F-ki) showed reduced incidence of neutrophil rTEM. Mechanistically, elevated vascular leakage promoted movement of interstitial chemokines into the bloodstream, a response that supported abluminal-to-luminal neutrophil TEM. Through development of an in vivo cell labeling method we provide direct evidence for the systemic dissemination of rTEM neutrophils, and showed them to exhibit an activated phenotype and be capable of trafficking to the lungs where their presence was aligned with regions of vascular injury. Collectively, we demonstrate that increased microvascular leakage reverses the localization of directional cues across venular walls, thus causing neutrophils engaged in diapedesis to reenter the systemic circulation. This cascade of events offers a mechanism to explain how local tissue inflammation and vascular permeability can induce downstream pathological effects in remote organs, most notably in the lungs.
Asunto(s)
Permeabilidad Capilar/inmunología , Microvasos/inmunología , Activación Neutrófila , Neutrófilos/inmunología , Migración Transendotelial y Transepitelial/inmunología , Animales , Permeabilidad Capilar/genética , Masculino , Ratones , Ratones Transgénicos , Microvasos/patología , Neutrófilos/patología , Migración Transendotelial y Transepitelial/genéticaAsunto(s)
Leucemia Linfocítica Crónica de Células B/patología , Microambiente Tumoral , Linfocitos T CD4-Positivos/patología , Ligando de CD40/inmunología , División Celular , Separación Celular , Técnicas de Cocultivo , Humanos , Inmunofenotipificación , Activación de Linfocitos , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacología , Células Tumorales Cultivadas/patologíaRESUMEN
Homing to distinct lymphoid organs enables chronic lymphocytic leukemia (CLL) cells to receive pro-survival and proliferative signals. Cytogenetic aberrations can significantly affect CLL cell compartmentalization. Trisomy 12 (tri12) defines a CLL subgroup with specific clinical features and increased levels of the negative prognostic marker CD49d, the α4-subunit of the integrin VLA-4, which is a key regulator of CLL cell homing to bone marrow (BM). Chemokine-induced inside-out VLA-4 activation, particularly via the CXCL12-CXCR4 axis, increases the arrest of various cell types on VCAM-1 presenting endothelium. Here, we demonstrate that high CD49d expression in tri12 CLL is accompanied by decreased CXCR4 expression. Dissecting functional consequences of these alterations, we observed that tri12 CLL cell homing to murine BM is not affected by CXCR4-CXCL12 blockage using AMD3100 or olaptesed pegol/NOX-A12. In line, CCL21-CCR7 rather than CXCL12-CXCR4 interactions triggered VLA-4-mediated arrests of tri12 CLL cells to VCAM-1 under blood flow conditions. Concordantly, in real-time kinetic analyses we found CCL21 but not CXCL12 being capable to induce inside-out VLA-4 conformational changes in this CLL subgroup. Our results provide novel insights into the peculiar clinico-biological behaviour of tri12 CLL and emphasize its specific chemokine and integrin utilization during pathophysiologically and therapeutically relevant interactions with the microenvironment.
Asunto(s)
Quimiocina CCL21/genética , Quimiocina CCL21/metabolismo , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Leucemia Linfocítica Crónica de Células B/genética , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Animales , Humanos , Ratones , Transducción de SeñalRESUMEN
Microenvironmental interactions are crucial for the survival and proliferation of chronic lymphocytic leukemia (CLL) cells. CD4+ T cells that express CD40 ligand (CD40L), along with other accessory immune and stromal cells within CLL lymph nodes, provide signals needed for activation and outgrowth of the tumor clone. Furthermore, correct positioning of CLL cells within lymphoid subcompartments is essential for the transmission of these supportive signals. Thereby, interstitial cell migration and adhesion events, influenced by activational stimuli, determine CLL cell localization. CD44 has been implicated in cell activation, migration, and tissue retention via binding to its extracellular matrix ligand hyaluronan (HA). In this study, we investigated the role of CD44-HA interactions for CLL positioning and interaction with supportive microenvironments in peripheral lymph nodes, focusing on its regulation via CD40L-dependent, T-cell-mediated activation of CLL cells. We found that hyaluronan triggered a robust CCL21-induced motility of resting CLL cells. However, CD40L stimulation promoted the firm, CD44-mediated adhesion of CLL cells to hyaluronan, antagonizing their motile behavior. N-linked glycosylations of CD44, particularly associated with the variant isoform CD44v6 after CD40L activation, seemed to facilitate hyaluronan recognition by CD44. We propose that the CD40L-CD40 signaling axis provides a stop signal to motile CLL cells within lymph node compartments by inducing high avidity CD44-HA adhesion. This might retain CLL cells close to T-cell stimuli and facilitate essential interactions with hyaluronan-bearing stromal cells, collectively promoting CLL cell proliferation and survival.
Asunto(s)
Antígenos CD40/inmunología , Quimiocina CCL21/inmunología , Ácido Hialurónico/metabolismo , Leucemia Linfocítica Crónica de Células B/inmunología , Antígenos CD40/metabolismo , Ligando de CD40/inmunología , Movimiento Celular/inmunología , Humanos , Ganglios Linfáticos/inmunología , Activación de Linfocitos/inmunología , Células Tumorales CultivadasRESUMEN
BACKGROUND: Celery (Apium graveolens) represents a relevant allergen source that can elicit severe reactions in the adult population. To investigate the sensitization prevalence and cross-reactivity of Api g 2 from celery stalks in a Mediterranean population and in a mouse model. METHODOLOGY: 786 non-randomized subjects from Italy were screened for IgE reactivity to rApi g 2, rArt v 3 (mugwort pollen LTP) and nPru p 3 (peach LTP) using an allergen microarray. Clinical data of 32 selected patients with reactivity to LTP under investigation were evaluated. Specific IgE titers and cross-inhibitions were performed in ELISA and allergen microarray. Balb/c mice were immunized with purified LTPs; IgG titers were determined in ELISA and mediator release was examined using RBL-2H3 cells. Simulated endolysosomal digestion was performed using microsomes obtained from human DCs. RESULTS: IgE testing showed a sensitization prevalence of 25.6% to Api g 2, 18.6% to Art v 3, and 28.6% to Pru p 3 and frequent co-sensitization and correlating IgE-reactivity was observed. 10/32 patients suffering from LTP-related allergy reported symptoms upon consumption of celery stalks which mainly presented as OAS. Considerable IgE cross-reactivity was observed between Api g 2, Art v 3, and Pru p 3 with varying inhibition degrees of individual patients' sera. Simulating LTP mono-sensitization in a mouse model showed development of more congruent antibody specificities between Api g 2 and Art v 3. Notably, biologically relevant murine IgE cross-reactivity was restricted to the latter and diverse from Pru p 3 epitopes. Endolysosomal processing of LTP showed generation of similar clusters, which presumably represent T-cell peptides. CONCLUSIONS: Api g 2 represents a relevant celery stalk allergen in the LTP-sensitized population. The molecule displays common B cell epitopes and endolysosomal peptides that encompass T cell epitopes with pollen and plant-food derived LTP.
Asunto(s)
Anticuerpos/inmunología , Antígenos de Plantas/inmunología , Apium/inmunología , Proteínas Portadoras/inmunología , Inmunización , Fragmentos de Péptidos/inmunología , Proteínas de Plantas/inmunología , Adolescente , Adulto , Alérgenos/química , Alérgenos/inmunología , Secuencia de Aminoácidos , Animales , Antígenos de Plantas/química , Antígenos de Plantas/metabolismo , Proteínas Portadoras/química , Línea Celular Tumoral , Niño , Estudios de Cohortes , Reacciones Cruzadas , Epítopos de Linfocito T/inmunología , Femenino , Fluoruros , Humanos , Hipersensibilidad/inmunología , Inmunoglobulina E/inmunología , Masculino , Metacrilatos , Ratones , Persona de Mediana Edad , Modelos Moleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Polen/inmunología , Poliuretanos , Conformación Proteica , Ratas , Adulto JovenRESUMEN
BACKGROUND: VLA-4 and CD38 predict a poor clinical outcome in chronic lymphocytic leukemia (CLL). We used CLL samples with discordant VLA-4/CD38 risk to address their individual roles in human bone marrow infiltration (BM), CLL cell homing to murine BM, and in supportive CLL cell-stromal cell interactions. METHODS: VLA-4, CD38, and Ki-67 expression was measured in CLL cells from peripheral blood (PB) and bone marrow (BM) aspirates. CLL BM infiltration rates, routinely determined by Pathology, were correlated to VLA-4 and CD38 expression. Short-term homing capacity of CLL cells was evaluated by adoptive transfer experiments. CLL cell viability and adhesion in stromal cell co-culture was determined. RESULTS: About 20% of CLL samples in our cohort displayed discordant VLA-4 and CD38 risk, with either high VLA-4 and low CD38 risk or vice versa. Using particularly such samples, we observed that VLA-4, and not CD38, was responsible for recirculation of CLL cells to murine BM. Human BM infiltration was also significantly higher in patients with high VLA-4 risk but not high CD38 risk. However, both molecules acted as independent prognostic markers. While both VLA-4 and CD38 expression were increased in BM-derived CLL cells, and VLA-4+ and CD38+ subpopulations showed enriched Ki-67 expression, VLA-4 did not contribute to CLL cell protection by stromal cells in vitro. CONCLUSIONS: Our data argue for a prominent role of VLA-4 but not CD38 expression in the homing of CLL cells to BM niches and in human BM infiltration, but only a limited role in their protection by stromal cells.
Asunto(s)
ADP-Ribosil Ciclasa 1/metabolismo , Médula Ósea/patología , Integrina alfa4beta1/metabolismo , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Infiltración Leucémica/patología , ADP-Ribosil Ciclasa 1/sangre , Animales , Apoptosis , Linfocitos B/inmunología , Médula Ósea/metabolismo , Adhesión Celular , Recuento de Células , Femenino , Humanos , Inmunohistoquímica , Integrina alfa4beta1/sangre , Antígeno Ki-67/metabolismo , Leucemia Linfocítica Crónica de Células B/sangre , Tejido Linfoide/patología , Masculino , Ratones , Factores de Riesgo , Células del Estroma/patología , Análisis de SupervivenciaRESUMEN
SCOPE: Celery represents a relevant cross-reactive food allergen source in the adult population. As the currently known allergens are not typical elicitors of severe symptoms, we aimed to identify and characterize a non-specific lipid transfer protein (nsLTP). METHODS AND RESULTS: MS and cDNA cloning were applied to obtain the full-length sequence of a novel allergenic nsLTP from celery stalks. The purified natural molecule consisted of a single isoallergen designated as Api g 2.0101, which was recombinantly produced in Escherichia coli Rosetta-gami. The natural and recombinant molecules displayed equivalent physicochemical and immunological properties. Circular dichroism revealed a typical α-helical fold and high thermal stability. Moreover, Api g 2 was highly resistant to simulated gastrointestinal digestion. As assessed by ELISA, thermal denaturation did not affect the IgE binding of Api g 2. Natural and recombinant Api g 2 showed similar allergenic activity in mediator release assays. Api g 2-specific IgE antibodies cross-reacted with peach and mugwort pollen nsLTPs. CONCLUSION: Based on our results, it can be anticipated that inclusion of recombinant Api g 2 in the current panel of allergens for molecule-based diagnosis will facilitate the evaluation of the clinical relevance of nsLTP sensitization in celery allergy and help clinicians in the management of food allergic patients.
Asunto(s)
Antígenos de Plantas/química , Antígenos de Plantas/genética , Antígenos de Plantas/inmunología , Apium/química , Proteínas Portadoras/química , Proteínas Portadoras/inmunología , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/inmunología , Tallos de la Planta/química , Secuencia de Aminoácidos , Animales , Reacciones Antígeno-Anticuerpo , Antígenos de Plantas/metabolismo , Apium/inmunología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Reacciones Cruzadas , Digestión , Hipersensibilidad a los Alimentos/inmunología , Humanos , Inmunoglobulina E/metabolismo , Datos de Secuencia Molecular , Proteínas de Plantas/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/metabolismo , Estabilidad Proteica , Estructura Secundaria de Proteína , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
Art v 3, the lipid-transfer protein (LTP) of Artemisia vulgaris pollen is a relevant allergen showing frequent cross-reactivity with homologues in other plants. Here we report the identification of four full-length Art v 3 sequences obtained by cDNA cloning using mass spectrometry-based sequencing. Two isoforms, Art v 3.0201 and Art v 3.0301 were expressed as soluble proteins in Escherichia coli Rosetta-gami B(DE3) pLysS using different expression systems. Purified natural and recombinant Art v 3 demonstrated similar secondary structures in circular dichroism analysis. All preparations showed high thermal stability but low resistance to gastric digestion with pepsin. Patient-specific IgE reactivity patterns to natural or recombinant isoallergens were observed among Art v 3-sensitized subjects. Using Immuno Solid-phase Allergen Chip (ISAC) assays, frequent cross-reactivity of Art v 3 with LTPs from peach and hazelnut was shown. The biological activity of both isoforms was comparable to the natural allergen in basophil release assays. The newly identified sequences provide the basis for recombinant mugwort LTP production enabling batch-to-batch reproducibility and thus ensuring high-quality products for diagnosis and therapy.