LRRK2) gene is associated with risk of familial and sporadic Parkinson’s disease (PD). To support clinical development of LRRK2 inhibitors as disease-modifying treatment in PD biomarkers for kinase activity, target engagement and kinase inhibition are prerequisite tools. In a combined proteomics and phosphoproteomics study on human peripheral mononuclear blood cells (PBMCs) treated with the LRRK2 inhibitor Lu AF58786 a number of putative biomarkers were identified. Among the phospho-site hits were known LRRK2 sites as well as two phospho-sites on human Rab10 and Rab12. LRRK2 dependent phosphorylation of human Rab10 and human Rab12 at positions Thr73 and Ser106, respectively, was confirmed in HEK293 and, more importantly, Rab10-pThr73 inhibition was validated in immune stimulated human PBMCs using two distinct LRRK2 inhibitors. In addition, in non-stimulated human PBMCs acute inhibition of LRRK2 with two distinct LRRK2 inhibitor compounds reduced Rab10-Thr73 phosphorylation in a concentration-dependent manner with apparent IC50’s equivalent to IC50’s on LRRK2-pSer935. The identification of Rab10 phosphorylated at Thr73 as a LRRK2 inhibition marker in human PBMCs strongly support inclusion of assays quantifying Rab10-pThr73 levels in upcoming clinical trials evaluating LRRK2 kinase inhibition as a disease-modifying treatment principle in PD.Genetic variation in the leucine-rich repeat kinase 2 ( LRRK2 cause clinical and pathological phenotypes of Parkinson’s disease (PD) that are undistinguishable from idiopathic PD5. Recent studies have shown that also common LRRK2 genetic variants are associated with risk of developing late onset PD11. Together, this suggests that LRRK2 signaling pathways may be central to the processes underlying both LRRK2 familial and sporadic late onset PD. One way to investigate the underlying role of LRRK2 in sporadic PD is to identify specific fingerprints reflecting LRRK2 kinase function and validate these in samples from patients suffering from idiopathic PD. In the past, efforts to perform such studies have been restricted by a limited understanding of LRRK2 function, its associated cellular signaling pathways as well as lack of good and selective pharmacological tools. Also, the predominant number of studies attempting to identify LRRK2 specific read-outs has been based on non-mammalian as well as non-physiological systems including various levels of exogenous over-expression of LRRK2 and other genetic manipulations16.Rare missense mutations in 22. Besides association to Parkinson’s disease LRRK2 genetic variation has also been associated with risk of multiple systems atrophy (MSA)23 and outside the CNS, LRRK2 has been associated with several diseases of the immune system28. Thus, we hypothesize that LRRK2 cellular mechanisms and signaling pathways are either conserved or exhibit high similarity between the central nervous system (CNS) and peripheral target tissues such as lung, kidney and immune cells. In support, LRRK2 phosphorylation at Ser910, Ser935, Ser955 and Ser973 used to assess LRRK2 enzymatic function is detected in all target tissues33. In rodents, phosphorylation of Ser910 and Ser935 is observed in brain, kidney, spleen and blood cells and phosphorylation levels correlate well between different tissues supporting the hypothesis that cellular mechanisms regulating LRRK2 function are conserved. Further, genetic ablation and pharmalogical inhibition of LRRK2 kinase function in rodents and non-human primates have identified LRRK2 kinase activity-dependent kidney and lung phenotypes38. Another example of a conserved LRRK2 fingerprint is the LRRK2-Ser1292 autophosphorylation site40 and more recent, in rodent transgenic models several small Rab GTPases have been confirmed as in vitro and in vivo substrates of LRRK241. These observations are of particular interest since in PD GWAS LRRK2 has previously been shown to genetically interact with PARK16 which is encoded by the RAB7L1 gene42. LRRK2 and phosphorylated LRRK2 have been observed in human blood, urine and CSF from healthy controls and PD patients45. In preclinical models LRRK2 inhibition with potent and selective LRRK2 kinase inhibitors has consistently been shown to reduce LRRK2 phosphorylation, autophosphorylation and now also substrate phosphorylation50. Collectively, LRRK2 appears to play an important physiological role outside the brain and its cellular fingerprints e.g. LRRK2 phosphorylation, LRRK2 auto phosphorylation and substrate phosphorylation seem well conserved between CNS and non-CNS cells. Still what has not been described are effects of LRRK2 inhibition on LRRK2 autophosphorylation and LRRK2 substrate phosphorylation in human primary cells expressing endogenous levels of LRRK2. In peripheral blood mononuclear cells (PBMCs) LRRK2 mRNA and protein expression can be induced by treatment with reagents known to initiate an immune response53. Thus, by applying PBMCs as a human LRRK2 model system, the goal of the underlying study was to identify novel LRRK2 kinase activity dependent substrates.In rodent and man LRRK2 is predominantly expressed in brain, spleen, lung, kidney and immune cells51. Presumably, this induction occurs under physiological conditions e.g. as part of the inflammatory response to viral infections54. Hypothesizing that LRRK2 signaling pathways are conserved between the CNS and the periphery we intended to identify novel LRRK2 kinase activity dependent substrates in PBMCs from healthy human individuals. To reduce variability between individual donors and to increase the likelihood of identifying LRRK2 kinase activity dependent substrates immune stimulated PBMCs were pursued in mass spectrometry studies. Firstly, studies were performed to ensure quality of PBMC sampling and culturing, that LRRK2 protein expression could be increased and that LRRK2 kinase inhibition could be obtained after LRRK2 inhibitor treatment. For that purpose three experimental conditions were assessed . In transiently transfected HEK293 cells the IC50 for Lu AF58786 on LRRK2 WT, the overactive variant G2019S and the inhibition resistant mutant A2016T56 were 12 nM, 19 nM and 93 nM, respectively . After a total of 72 hrs in culture the PBMC’s were harvested, pelleted and fresh-frozen on dry-ice. An aliquot of PBMCs was used to assess PBMC yield and viability after culturing. The average viability was above 95% and the average yield after culturing was ~6 × 106 cells pr. sample  and PBMC yield and viability was evaluated. As for the optimization studies there were no significant differences in PBMC yield or viability between subjects. The yield was ranging between 0.9–1.3 × 10ple Fig. . There wple Fig. . A commoTo enable quantitative mass spectrometry of human PBMCs chemical labeling with mTRAQ reagents (ABSciex) was applied subsequent to PBMC pellet extraction. Upon cell lysis of the individual cell preparations and tryptic protein cleavage peptide mixtures were desalted and peptides were then differentially isotope-labeled with mTRAQ Δ0 (light), mTRAQ Δ4 (medium) or mTRAQ Δ8 (heavy) reagents. For all samples mTRAQ labeling efficiencies were determined and found to be higher than 95% throughout the study 72.Several LRRK2 phosphorylation sites were among the regulated phosphoproteins. More than two-fold reductions were observed for LRRK2-pSer910, -pSer935, -pSer955 and -pSer973 sites. Fig. . Notablyin vitro kinase assays41. LRRK2 mediated phosphorylation of these Rab family proteins occurs in the conserved switch II region of the protein which is involved in GTP hydrolysis and binding to regulatory proteins73. Notably, our phosphoproteomics dataset comprises more than 40 distinct class I phosphorylation sites on 25 different Rab GTPase proteins that were quantified in at least 5 biological replicate experiments. This data set also comprises information for the conserved regulatory serine or threonine residue in the switch II domain of 10 distinct Rab proteins. Our statistical analyses revealed that Thr73 on Rab10 and Ser106 on Rab12 show an approximately 2-fold reduced level of phosphorylation upon treatment with the LRRK2 inhibitor while all other Rab GTPase proteins such as Rab1B, Rab7A and Rab8A were not affected in their level of phosphorylation . Titer determination of the affinity-purified antibodies using phospho and non-phospho peptides of Rab10 and Rab12 found that both antibodies were recognizing the phosphorylated peptides of Rab10 and Rab12  from transgenic and wild type animals, Rab10 in lung and spleen from wildtype mice and Rab12 in brain from G2019S transgenic mice as well as in heterologous expression systems such as HEK293 cells overexpressing LRRK2 variantset al. suggesting that several Rab GTPases including Rab10 and Rab12 are LRRK2 kinase substrates41. Rab10 and Rab12 belong to the family of small Rab GTPases. These proteins are known to be master regulators of intracellular vesicular trafficking75. Besides Rab10 and Rab12 also Rab8A and Rab7L1 were proposed as LRRK2 substrates by Steger et al.41. This is of particular interest since Rab7L1 is part of the LRRK2 interactome76, Rab7L1 deficient mice show kidney abnormalities60 similar to those observed in LRRK2 KO mice and in GWAS single nucleotide polymorphisms in the gene encoding the small Rab GTPase RAB7L1 have been shown to genetically interact with the LRRK2 loci to modulate risk of Parkinson’s disease77. Another PD-associated kinase PINK1 is found to mediate indirect phosphorylation of Rab8A at position Ser-111 thereby regulating Rab8A activation78 and accumulating evidence also links the α-synuclein biology to several Rab GTPases here including Rab8A87. Thus, involvement of Rab GTPase and intracellular vesicular trafficking may prove to underlie neurodegenerative mechanisms associated with some aspects of α-synuclein pathology in PD.Our findings in cultured human PBMCs also validate recent observations obtained in animal models and heterologous expression systems by Steger 88. It is localized to early endosomes where it regulates vesicular recycling of cargo molecules to and from the plasma membrane92. In macrophages Rab10 expression is induced upon TLR4 stimulation suggesting a functional role for Rab10 in immune stimulated mononuclear peripheral blood cells93. Rab10 is involved in membrane transport in kidney cells95, has been functionally coupled to autophagic processes and interestingly, genetic ablation of RAB10 leads to accumulation of lipid droplets in hepatocytes96. LRRK2 is not expressed in the liver; however, the latter observation is not unlike observations of lipid droplet accumulations in the kidneys of LRRK2 KO rats and mice suggestive of Rab10 playing a functional role in LRRK2 kinase activity dependent mechanisms97. This study shows that Rab10 phosphorylation is dependent on LRRK2 kinase activity in human PBMCs. Further, compared to LRRK2 WT the disease variants G2019S and R1441C exhibits increased pThr73 levels in HEK293 cells  Ltd . PFE-360 was synthesized at H. Lundbeck as described in patent application US2014/005183 (Example 217). The structure of compound A (Cmpd A) cannot be disclosed at this stage. The structure will be published in an upcoming publication.All studies on human donor blood samples were carried out in accordance with guidelines and regulations of Danish legislation. Blood was donated at the Danish Bloodbank at Hvidovre Hospital . At the donation site, all human donor blood samples were fully anonymized and informed consent was obtained from all human subjects. According to Danish legislation, experiments performed on anonymized human bio samples do not require approval from a local ethics committee. All subsequent experiments were performed at Lundbeck  according to guidelines and regulations of Danish legislation.Donor blood from human healthy subjects were collected at the Danish Bloodbank at Hvidovre Hospital in 9 mL K2-EDTA tubes (Vacutainer BD 367525). After blood collection, tubes were immediately turned-over several times to mix blood and anticoagulant. The samples were shipped at controlled temperature ranging from 15–25 °C. Receipt of samples was within 2 h following blood collection.PBMC were isolated from fresh blood collected within a maximal time of 4 hours after blood collection. Leucosep tubes (Greiner 227290) was added 15 ml Ficoll  and spun 30 s at 1000 × g. Blood was diluted 1:1 using D-PBS 1X (Gibco 14190), transfered to the Leucosep and centrifuged at 800 g for 15 min without brake. The upper layer corresponding to the diluted plasma was removed and the PBMC layer collected using a pipette or by pouring into a clean BD Falcon 50 ml tube. Cells were re-suspended and washed four times with 10 ml of D-PBS 1X (Gibco 14190) followed by centrifugation at 250 g for 10 min with brake at room temperature. The supernatant was discarded and the cells re-suspended in Complete medium equilibrated to 37 °C. The number of dead and living cells were counted on a Nucleocounter and used to calculate percent viability.6 cells in 10 cm petri dishes (Costar 3262) in RPMI-1640 media (Gibco 61870–010) supplemented with 10% non-heat inactivated FBS, 5 ml Antimycotic (Sigma cat:5965) with or without an immune stimulation cocktail consisting of 400 nM phorbol 12-myristate 13-acetate (PMA) and 100 ng/ml interferon-γ (INF-γ). After 48 hrs in culture the cultures were added a fifth of the volume media containing 5x the final concentration of the respective LRRK2 inhibitor with or without PMA and interferon-γ. For the PBMC optimization studies the experimental conditions were as follows: (1) control – plated, un-induced and untreated (n = 10); (2) immune stimulated - induced with PMA/INFγ for 72 hrs (n = 10) and (3) immune stimulated and compound treated - induced with PMA/INF-γ for 72 hrs and treated with the potent and selective LRRK2 kinase inhibitor Lu AF58786 for the last 24 hrs (n = 10). Approximately, 400 µg of protein from each individual donor was needed in order to run the full PBMC stimulation and inhibition study with subsequent global proteome and phosphoproteome (PhosphoScout®) analyses. After 72 hrs in culture the PBMC’s were harvested, pelleted and fresh-frozen on dry-ice. An aliquot of PBMCs was used for assessment of PBMC yield and viability. For the PBMC proteomic studies the following experimental conditions were used: (1) immune stimulated - induced with PMA/INFγ for 72 hrs ; (b) immune stimulated and compound treated - induced with PMA/INF-γ for 72 hrs and treated with the LRRK2 kinase inhibitor Lu AF58786 for the last 24 hrs .Freshly isolated PBMCs were plated at a density ranging from 7–20 × 10in vitro target engagement and kinase selectivity, respectively  where LRRK2 target engagement and broad kinase profiling was performed using KiNativ™ procedures. Live cell treatment of human peripheral blood mononuclear cells (PBMCs) at two compound concentrations (1 µM and 0.1 µM) were used to evaluate and compare 2 and shipped to Evotec on dry-ice. Subsequent sample preparation for MS analysis was executed by Evotec Munich. Cell lysis was performed in ice-cold lysis buffer ) and Complete protease inhibitor cocktail tablets (Roche). Cell extracts were sonicated on ice and the cell debris was removed by centrifugation before protein concentrations were determined .Upon the respective treatments cells were washed with cold PBS, snap-frozen in liquid NEach sample was reduced with 10 mM dithiothreitol for 30 min and thereupon alkylated in the presence of 55 mM iodoacetamide for 30 min in the dark. Subsequently, endoproteinase Lys-C (Wako) was added at an enzyme-to-substrate ratio of 1:100 and incubated for 2 h at room temperature. Samples were thereafter diluted 1:4 with 20 mM Tris-HCl pH 8.2 before adding trypsin (Promega) at an enzyme-to-substrate ratio of 1:100 followed by overnight incubation. The resulting peptide mixtures were acidified by the addition of trifuoroacetic acid (TFA) to a final concentration of 0.5% and subsequently desalted using C18 Sep-Pak columns . Peptides were eluted with 50% acetonitrile, 0.5% acetic acid, and aliquoted into the respective fraction that was utilized for the individual replicate experiment and/or for the pooled reference sample. Aliquoted samples were snap-frozen in liquid nitrogen and lyophilized.et al.98. Briefly, peptides were reconstituted in 20 mM ammonium formate , loaded onto an XBridge C18, 200 × 4.6 mm analytical column (Waters) operated with the Äkta Explorer system  and separated by applying a segmented gradient increasing the acetonitrile concentration from 7% to 30% buffer B (buffer A supplemented with 80% acetonitrile) over 15 min followed by a 5 min gradient to 55% over 5 min. For each individual experiment the collected peptide fractions were then combined in a non-linear way to generate 15 samples with equal peptide amounts. Fractions were frozen in liquid nitrogen, lyophilized, reconstituted in 0.1% TFA and desalted using C18 Sep-Pak columns . An aliquot of each fraction corresponding to approximately 5 μg peptide starting material was removed for global proteome analysis. The large remainder of each fraction was frozen in liquid nitrogen and lyophilized and subsequently applied to phosphopeptide enrichment. Enrichment of phosphopeptides was performed according to a protocol by Steven Carr and coworkers99. Briefly, peptides of each fraction were reconstituted in IMAC loading buffer (80% acetonitrile containing 0.1% TFA) to reach a final concentration of approximately 5 mg/ml. To generate the IMAC-resin Ni ions of Ni-NTA Superflow Agarose Beads (Qiagen) were removed by incubation with 100 mM EDTA and subsequently replaced by Fe3+ ions. The resin was thereupon washed with H2O and reconstituted in a 1:1:1 mix of acetonitrile, methanol and 0.01% acetic acid. To each sample 10 μl of equilibrated IMAC resin was added and incubated for 30 min at 25 °C and 1,400 rpm in a Thermomixer (Eppendorf). This slurry was then loaded onto in-house build C18 StageTips columns100. Subsequently beads were washed with IMAC loading buffer and phosphopeptides were eluted onto the C18 material by washing twice with a 500 mM K2HPO4 solution. The C18-bound phosphopeptides were washed with 0.1% formic acid, eluted with 50% ACN, 0.1% FA, concentrated in a VacufugeTM (Eppendorf) and reconstituted in 0.1% FA before MS analysis.All samples were then chemically labeled with the respective mTRAQ isotopic variants (ABSciex) according to the manufacturer’s instructions. The labeling efficiencies were determined for each sample and found to be above 95% for all samples  experiments were performed on a Q Exactive mass spectrometer (Thermo Fisher Scientific) equipped with an Easy nLC-1000 UPLC system (Thermo Fisher Scientific). Phosphopeptide-enriched samples were loaded with an auto sampler onto a 40 cm fused silica emitter (New Objective) packed in-house with reversed phase material  at a maximum pressure of 950 bar. The bound peptides were eluted over 125 min run time and sprayed directly into the mass spectrometer using a nanoelectrospray ion source (ProxeonBiosystems). The mass spectrometer was operated in the data dependent mode to automatically switch between MS full scans at a resolution R = 70,000 (at m/z  200) with a target value of 3,000,000 counts (max. Injection time = 45 ms) and MS/MS fragmentation scans at R = 35,000 and target value of 200,000 ions (max. Injection time = 120 ms). The ten most intense peptide ions were selected higher-energy collisional dissociation (HCD).Mass spectrometric analyses for the accompanying global proteome analysis was performed with a LTF-Orbitrap Velos (Thermo Fisher Scientific) equipped with an Easy nLC-1000 UPLC system (Thermo Fisher Scientific). Samples were loaded as described above.The mass spectrometer was operated in the data dependent mode to automatically switch between MS full scans at a resolution R = 60,000 (at m/z 200) with a target value of 1,000,000 counts (max. Injection time = 500 ms). The fifteen most intense peptide ions were selected for collision induced fragmentation and the resulting fragmentation spectra were recorded in the linear ion trap at a target value of 5000 ion counts (max. Injection time = 25 ms). The resulting fragmentation spectra were recorded in the linear ion trap.101. The maximum mass deviations allowed for MS and MS2 peaks were 6 ppm and 0.5 Da for data acquired with the LTQ-Velos and 4.5 ppm and 20 ppm for data acquired with the Q Exactive. Carbamidomethylation of cysteine was set as a fixed modification and oxidation of methionine, protein N-terminal acetylation were allowed as variable modifications. In addition, for phosphopeptide-enriched samples also phosphorylation of serine, threonine and tyrosine residues were allowed as variable modifications. All peptides were required to have a minimum peptide length of seven amino acids and a maximum of two missed cleavages and three labeled amino acids were allowed. The false discovery rate (FDR) for protein and peptide identifications of set to 1% and the match between runs option was enabled.All raw files acquired in this study were collectively processed with the MaxQuant software suite (version 1.5.3.31) for peptide and protein identification and quantification using a human uniprot databases (version 09 2015)102. For the comparison of cmpd versus DMSO treated PBMC the ratios were calculated via the respective pooled reference sample (light mTRAQ label) for each individual replicate experiment and a hypothesis test was conducted to identify significantly regulated phosphorylation sites. For these analyses, only phosphorylation sites that were quantified in at least five out of the eight replicate experiments were used. Significantly regulated sites were identified with the mean rank test using an estimated FDR of 5%103. Similar analyses were performed with the global proteome data to identify proteins that became significantly regulated upon the different treatments.The resulting list of phosphorylation sites exported from the MaxQuant software was filtered for class-I sites, i.e. phosphorylation sites that could be located with high confidenceEnrichment analyses of gene ontology (GO) terms and KEGG pathways were performed by applying Fisher’s exact test and utilizing all proteins with significantly altered class I phosphorylation sites as input.104. Only high confidence interactions (confidence score >0.7) were considered. Proteins without any interactions satisfying the above criteria were removed from the network.For a network analysis all proteins with significantly regulated phosphorylation sites (FDR <5%) were mapped onto the STRING protein-protein network version 10Phospho-specific Rab10 and Rab12 antibodies targeted against Thr73 and Ser106, respectively, were generated at Genscript using a customized “Phospho-specific Polyclonal Antibody Package”. For each antibody project four rabbits were immunized at least three times using either Rab10-Thr73 or Rab12-Ser106 phosphopeptides coupled to a KLH conjugate. The sequences used for immunization were ERFH(pT)ITTSYYRC and ERFN(pS)ITSAYYRSAKC for Rab10 and Rab12, respectively. Subsequent to the 3rd immunization titer determination of phosphopeptide and non-phosphopeptide affinities guided the selection of rabbits for small-scale bleed, affinity purification as well as indirect ELISA determination of antibody specificity and selectivity. Coating antigens used for the indirect ELISA were the phosphopeptides or non-phosphopeptides. The antigens were diluted in Phosphate Buffered Saline (PBS) at pH 7.4 and used in a coating concentration and volume of 4 μg/ml and 100 μl/well. The secondary antibody used for quantification was a peroxidase-conjugated goat anti-rabbit IgG. Finally, rabbits proceeded towards final boost, bleed and affinity purification of the antibodies. The ELISA results for the affinity purified Rab10-pThr73 (#5981) and the Rab12-pSer106 (#5919) antibodies are listed in Supplementary Tables Plasmids encoding various LRRK2 and Rab GTPase protein variants were transiently expressed in HEK293 cells using Lipofectamine®2000 according to the manufacturer’s instructions .50 of Lu AF58786 was determined using a cell-based quantitative immunocytochemistry 96-well ICW assay based on LRRK2-Ser935 phosphorylation using theOdyssey CLx near infra-red technology . HEK293 cells grown in clear-bottom 96-well plates were transfected with either wild type LRRK2, kinase overactive variant G2019S, inhibition-resistant mutant A2016T or mock, respectively. One hour prior to cell fixation plates were pre-incubated with various concentrations of Lu AF58786 to inhibit LRRK2-Ser935 phosphorylation. Subsequently, the plates were processed as previously described105.Cell-based IC6 cells/well in 6-well plates pre-coated for 1 hr at 37 °C with poly-l-lysin. Subsequent to the transient transfection HEK293 cells were harvested using 1 mL cold Phosphate Buffered Saline (PBS) buffer  and subsequently centrifuged at 800 g for 2 minutes. Cell pellets were resuspended and solubilized in 100 µL lysis buffer , pH 8.0) on ice for 20 minutes and then centrifuged at 20,000 g for 30 minutes at 4 °C. The solubilized crude cell lysate was size mobility separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). LRRK2 was separated on a 3–8% Tris-Acetate gel  and Rab10 and Rab12 were separated using a NuPAGE® Novex 4–12% Bis-Tris Gel . An amount of 10 µg total protein was loaded in each well. Proteins were transferred to immobilon-FL PVDF membranes . Membranes were incubated with primary antibodies overnight at 4 °C: mouse monoclonal [N241A/34] anti-LRRK2 antibody , rabbit monoclonal [UDD2 10(12)] anti-pS935-LRRK2 antibody , mouse anti-HA monoclonal antibody [clone HA-7] , rabbit anti-Rab10-pThr73 polyclonal [#5981]  and rabbit anti-Rab12-Ser106 polyclonal antibody [#5919] . Subsequently, the membranes were washed and incubated with secondary antibodies for 1 hour at room temperature: Anti-rabbit IgG F(c) (GOAT) antibody IRDye® 800CW Conjugated  and anti-mouse Alexa Fluor® 680 Goat anti-mouse IgM . The proteins were visually detected by infrared imaging using Odyssey CLx . Membranes were scanned and band intensities were quantified using the Image Studio version 3.1.4 .HEK293 cells were plated at a density of 1 × 106 cells/ml in PBS. Donor samples were treated with the LRRK2 inhibitor PFE-360 for one hour and the cells were hereafter lysed by adding 1/10 volume lysis buffer  containing phosphatase and protease inhibitor cocktail . The lysates from human PBMCs were subjected to SDS-PAGE and Western Blotting procedures as described above.PBMCs were isolated using the density gradient medium, Lymphoprep™ from Stemcell Technologies  following their standard protocol. Briefly, 4 ml blood was diluted with 4 ml PBS and applied at the SepMate™ tubes (Stemcell Technologies) and spun at 1200xg for 20 min at RT in the centrifuge without brakes. The PBMC layer was collected and spun at 600xg for 8 min and washed two times in PBS by spinning at 600xg between each wash. After the last wash, the cells were counted and the cellular concentration was adjusted to 50 × 10Data and statistical analyses were performed using Prism 5  Data were analyzed by either 1-way or 2-way analysis of variance (ANOVA) or by t-test. Post-hoc tests following ANOVAs were conducted using Dunnett’s or Holm-Sidak’s multiple comparisons test. Two-tailed levels of significance were used and p < 0.05 was considered statistically significant.Supplementary Data FileSupplementary File 1Supplementary File 2


Ambrosia artemisiifolia) and mugwort (Artemisia vulgaris) are the major cause of pollen allergy in late summer. Allergen-specific lymphocytes are crucial for immune modulation during immunotherapy. We sought to generate and pre-clinically characterise highly immunogenic domains of the homologous pectate lyases in ragweed (Amb a 1) and mugwort pollen (Art v 6) for immunotherapy.Ragweed  and Art v 6 (Art v 6α) and a hybrid molecule, consisting of both domains, were designed, expressed in in vitro allergenic activity of the Amb a 1 and Art v 6 domains and the hybrid were either greatly reduced or abolished. The recombinant proteins induced T cell proliferative responses comparable to those of the natural allergens, indicative of retained allergen-specific T cell response. Mice immunisation with the hypoallergens induced IL-4, IL-5, IL-13 and IFN-γ production after antigen-specific in vitro re-stimulation of splenocytes. Moreover, murine IgG antibodies that inhibited specific IgE binding of ragweed and mugwort pollen allergic patients were detected.The IgE binding capacity and Accumulation of T cell epitopes and deletion of IgE reactive areas of Amb a 1 and Art v 6, modulated the immunologic properties of the allergen immuno-domains, leading to promising novel candidates for therapeutic approach. Ambrosia artemisiifolia), which represents one of the most prominent seasonal allergen sources across the United States, Canada and parts of Europe thymidine (0.5 μ Ci/well) after 16 h by scintillation counting as described thymidine uptake as described [Art v 6- and Amb a 1–specific T-cell lines (TCLs) were established from PBMCs in triplicates by using nArt v 6 or nAmb a 1 (2 μg/well), as the initial stimulus. The proliferative responses of TCL to nAmb a 1, nArt v 6, Amb a 1F, Art v 6α or hybrid (25 pmol) were assessed after 48 h by [escribed .Female BALB/c mice  8–10 weeks old were immunised subcutaneously with 5 μg antigen adsorbed to Alugel-S  or PBS given as two 50-μL subcutaneous injections administered bilaterally in the lumbar region and boosted on days 23, 38 and 52. Sera were collected on days 0, 31 and 64. Six animals per group were tested.1 and IgG2a was analysed by ELISA as described [1 and IgG2a alkaline phosphatase  were added to detect bound IgG and the reaction developed with p-nitrophenyl phosphate (PNPP) (Sigma-Aldrich). Absorption was measured at 405 nm. For inhibition assay coated antigen was incubated with mouse serum (dilution 1:20) for 16 h at 4°C. Individual human sera (n = 5) were added in a 1:10 dilution, and bound IgE was detected as described for direct ELISA.Serum IgGescribed . In brieRBL-H2H3 cells were incubated with a pool of 6 mice sera (1/60) for 2 h at 37°C. After adding 0.3 μg/ml of each allergen, degranulation of RBL cells was induced and β-hexosaminidase activity in the supernatant was analysed as previously described .ad libitum. Animals were kept in groups of five per IVC module at 20–24°C and a relative humidity of 55%. Bedding and water was changed weekly. The light/dark cycles were set in 12 h intervals with a 30 min dim period from 0–200 lux and vice versa. Blood samples were taken by punctuation of the vena facialis. For analyses of splenocytes animals were sacrificed via cervical dislocation.Antigen-specific IL-4, IL-5, IL-10, IL-13 or IFN-γ-secreting splenic lymphocytes were detected by ELISPOT  according to the manufacturers’ instructions. Animal experiments were conducted in accordance with EU guidelines 86/609/EWG and national legal regulations (TVG 2012). This study was approved by the Austrian Ministry of Science, Ref. II/3b (Gentechnik und Tierversuche), permission number BMWF-66-012/0010-II/3b/2013. Mice were kept in individually vented IVC modules in the SPF  animal facilities at the University of Salzburg. Food and water was given P values < 0.05 were considered significant.Data were analyzed by GraphPad Prism version 5 . Statistical analysis was performed using ANOVA and Bonferroni post test. E. coli as C-terminally 6x His-tagged fusion protein .Amb a 1α (rAmb a 1 residues 174–397), Art v 6α (rArt v 6 residues 173–396), and a hybrid molecule containing rAmb a 1 residues 174–373 at the N-terminus and rArt v 6 residues 173–370 at the C-terminus, linked by 2 glycine residues, were codon-optimized and produced in Fig 2A).The purified proteins showed a mixture of α-helical and β-sheet secondary structure elements, and a folded state as determined by far-UV circular dichroism .Compared to purified natural allergens, each of the Amb a 1α and Art v 6 α and hybrid molecules were virtually not recognized by specific IgE from 30 ragweed/mugwort-allergic patients (P< 0.001) . Inhibition of IgE binding to nAmb a 1 by preincubation with nArt v 6 ranged from 45 to 65%, whereas inhibition of IgE binding to Art v 6 by preincubation with Amb a 1 ranged from 60 to 80%. Compared to purified natural allergens, the domains’ inhibitory capacity was significantly (P<0.001) reduced at 50 pmol , 0.5 pmol and 0.005 pmol (results not shown).Furthermore, cross-inhibitory experiments with coated natural and recombinant proteins as inhibitors were performed. Self-inhibition of IgE binding by nAmb a 1 and nArt v 6 reached 90.5% and 82%, respectively  .The low IgE-binding capacity of the recombinant domains was confirmed by the lack of induction of mediator release even at the highest concentration (10 μg/ml). The natural proteins showed biological activity between 0.1 μg/ml and 10 μg/ml with a maximum mediator release of 60–70%  and purified natural allergens.Comparable proliferative responses in human PBMCs isolated from 10 ragweed/mugwort pollen-allergic donors were induced by equimolar amounts of the domains . Amb a 1α, Art v 6α and the hybrid molecule induced responses in 7, 6 and 8 of 8 nAmb a 1-specific TCL, respectively , and 5, 3, and 5 of 9 nArt v 6-specific TCL, respectively , confirming that the relevant Amb a 1/Art v 6 T cell epitopes are located in the alpha chain domains.Similarly, nAmb a 1- and nArt v 6-induced T cell lines (TCL) established from 14 ragweed/mugwort pollen-allergic patients and re-stimulated with natural or recombinant protein (25 pmol), displayed proliferation responses that were not significantly different between the natural and the constructs  were measured by ELISA. The domains induced IgG1 and IgG2a antibodies against nAmb a 1 and nArt v 6 .To investigate the immunogenic properties of the recombinant proteins, mice were immunised with the natural or recombinant proteins, and the levels of serum IgGFig 5A). IgG2a levels were comparable between proteins . Lack of Bet v 1-specific IgG1 and IgG2a was observed (results no shown).Cross-reactive IgG response was similar between the domains and nAmb a 1 but significantly stronger when compared to nArt v 6. Interestingly, the hybrid induced cross-reactive nArt v 6 IgG antibodies that were significantly higher than those induced by nAmb 1-immunised mice (P<0.001) .Degranulation by biologically active anti–Amb a 1 or Art v 6–specific IgE antibodies was confirmed by means of RBL-2H3 assay in sera of 6 mice immunized with nAmb a1, nArt v 6, Amb a 1α, Art v 6α or hybrid. The natural proteins showed biological activity with a maximum mediator release of 90% by nAmb a 1 and 82% by nArt v 6. Moreover, a significantly reduced capacity to induce mediator release by the variants compared to the natural proteins was also confirmed in our mouse model .Mouse IgG antibodies induced by the recombinant domains partly inhibited the IgE binding of 5 ragweed/mugwort pollen-allergic patients’ sera IgE to nAmb a 1 (30–35% mean inhibition) and nArt v 6 (17–21%), indicative of the presence of conserved antibody epitopes on the alpha domains . Recombinant proteins induced similar IL-5 production compared to the natural allergens . Only Amb a 1α immunisation induced significantly higher IFN-γ levels than the natural allergens. Whereas the hybrid molecule induced similar IL-4 levels compared to the natural molecules, both Amb a 1α and Art v 6α immunisation induced significantly higher levels. Low and comparable levels of IL-13- or IL-10-secreting T cells were detected, whereas immunization with nAmb a 1 failed to induce IL-10-secreting cells (P<0.05) . These results demonstrate that the alpha domains are able to evoke a TH1/TH2 type immune response.Splenocytes from immunised mice re-stimulated with each of the 5 proteins were evaluated for cytokine production by ELISPOT  was observed, which could be an indication that the murine immune response towards PL allergens is quite distinct from the human allergic response. These results could however be biased by the fact that an adjuvant was used in the murine immunization schedule resulting in differences in allergen presentation or processing by mice and humans, which are exposed in a completely different manner ,24. ConsBesides epitope modifying effects the adjuvant ALUM may also bias the subsequent immune response on the T cell level. Here were measured a TH2-polarizing effect of ALUM, indicated by the production of IL-4, IL-5 and IL-13. Analyses of murine splenocytes revealed the induction of TH1 cells producing IFN- γ by the recombinant proteins. In other words, a mixed TH1/TH2 immune response was elicited against Amb a 1α, Art v 6α, and hybrid compared to a clear TH2-driven response induced by the natural proteins.vice versa. However, with our study results we suggest that a single allergen derivative might be sufficient for a concomitant treatment strategy of PL mediated allergies form the Asteraceae family.Concomitant allergies to ragweed and mugwort pollen are a well-recognized problem in areas where both plants are endemic, where it is difficult to distinguish between co-sensitization and cross-reactivity . Amb a 1in vitro allergenicity. These results in humans correlated with results gained from a murine immunization model where Amb a 1α, Art v 6α, and hybrid were compared to wildtype allergens. Moreover, the single domains seemed sufficient to induce robust levels of IgG antibodies cross-reactive with the full-length WT allergens. Thus, based on our results we successfully generated candidates for AIT to redirect the allergic immune response supporting the idea of using only the alpha chain domain of Asteraceae PL allergens.In summary, the accumulation of T cell epitopes and the deletion of IgE reactive areas of the PL allergens Amb a 1 and Art v 6 resulted in the generation of highly immunogenic domains of both proteins. This led to the development of a hybrid molecule consisting of the combination of Amb a 1 and Art v 6 C-terminal regions. The domains were able to induce lymphoproliferative responses in ragweed/mugwort pollen-allergic patients´ PBMCs and displayed low IgE reactivity and S1 Dataset(A) Superimposed far-UV CD spectra of Amb a 1α, Art v 6α and hybrid at 0.1 mg/ml in 10 mM sodium phosphate buffer (pH 7.4). Data are presented as residue molar ellipticity. (B) Dataset information corresponding to specific IgE reactivity of 30 patients to natural and recombinant proteins tested by ELISA (C) Dataset information corresponding to inhibition ELISA of serum IgE from 5 allergic donors.(XLSX)Click here for additional data file.S2 DatasetInduction of mediator release by nAmb a 1, nArt v 6, Amb a 1α, Art v 6α and hybrid (0.0001–10 μg/ml) using RBL-2H3 cells and sera of 5 individual patients.(XLSX)Click here for additional data file.S3 Dataset(A). Antigen-specific T-cell lines (TCLs) were established from PBMCs by using nArt v 6 or nAmb a 1 as the initial stimulus. nAmb a 1- (B) and nArt v 6-(C)- specific TCL proliferative responses to nAmb a 1, nArt v 6, Amb a 1α, Art v 6α or hybrid were assessed. Results were expressed as stimulation indices (SI).PBMCs from 10 individual patients were stimulated with 62 pmol (a) of nAmb a 1, nArt v 6, Amb a 1α, Art v 6α and hybrid (XLSX)Click here for additional data file.S4 Dataset1(A) and IgG2a(B) antibodies levels against nAmb a 1 and nArt v 6 were analyzed by ELISA. (C) Mediator release from RBL-2H3 cells sensitised with murine serum pools (n = 6) from mice immunized with nAmb a 1, nArt v 6, Amb a 1α, Art v 6α or hybrid and stimulated with natural or recombinant proteins (0.3 μg/ml. (D) nAmb a 1 or nArt v 6-coated plates were preincubated with pooled sera from mice immunized with nAmb a 1, nArt v 6, Amb a 1α, Art v 6α or hybrid and the percentage of inhibition of serum IgE binding of 5 allergic patients to nAmb a 1 or nArt v 6 was determined by ELISA.BALB/c mice (n = 6/group) were immunized 4 times with natural or recombinant proteins (5 μg/ml) and the serum specific IgG(XLSX)Click here for additional data file.S5 Dataset of splenocytes from animals (n = 6/ group) immunized with nAmb a 1, nArt v 6, Amb a 1α, Art v 6α or hybrid and stimulated with natural or recombinant proteins are expressed as cytokine-secreting cells per 2x105 spleen cells.ELISPOT analyses (XLSX)Click here for additional data file.


Previously, we reported that cAMP/PKA signaling is involved in GPER-mediated coronary relaxation by activating MLCP via inhibition of RhoA pathway. In the current study, we tested the hypothesis that activation of GPER induces coronary artery relaxation via inhibition of RhoA/Rho kinase pathway by cAMP downstream targets, exchange proteins directly activated by cAMP (Epac) as well as PKA. Our results show that Epac inhibitors, brefeldin A , or ESI-09 (20 μM), or CE3F4 (100 μM), all partially inhibited porcine coronary artery relaxation response to the selective GPER agonist, G-1 (0.3–3 μM); while concurrent administration of BFA and PKI (5 μM), a PKA inhibitor, almost completely blocked the relaxation effect of G-1. The Epac specific agonist, 8-CPT-2Me-cAMP , induced a concentration-dependent relaxation response. Furthermore, the activity of Ras-related protein 1 (Rap1) was up regulated by G-1 (1 μM) treatment of porcine coronary artery smooth muscle cells (CASMCs). Phosphorylation of vasodilator-stimulated phosphoprotein (p-VASP) was elevated by G-1 (1 μM) treatment, but not by 007 (50 μM); and the effect of G-1 on p-VASP was blocked by PKI, but not by ESI-09, an Epac antagonist. RhoA activity was similarly down regulated by G-1 and 007, whereas ESI-09 restored most of the reduced RhoA activity by G-1 treatment. Furthermore, G-1 decreased PGF2α-induced p-MYPT1, which was partially reversed with either ESI-09 or PKI; whereas, concurrent administration of ESI-09 and PKI totally prevented the inhibitory effect of G-1. The inhibitory effects of G-1 on p- MLC levels in CASMCs were mostly restored by either ESI-09 or PKI. These results demonstrate that activation of GPER induces coronary artery relaxation via concurrent inhibition of RhoA/Rho kinase by Epac/Rap1 and PKA. GPER could be a potential drug target for preventing and treating cardiovascular diseases. G-protein-coupled estrogen receptor 1 or GPER is emerging as therapeutic target for the treatment of CVD , 3, and The newly-discovered target of cAMP, exchange proteins directly activated by cAMP (Epac), has been revealed to be a novel downstream mechanism for cAMP to govern signaling in the cardiovascular system and other tissues , 12.Its 3, 1.2 KH2PO4, 1.2 MgCl2, 1.8 CaCl2, 11.5 glucose, pH 7.2, bubbled with 95% O2−5% CO2 (pH 7.4) at 37°C. One wire was connected to a force-displacement transducer and the other to a stationary micrometer. The equilibration time for the preparations in Krebs-Henseleit buffer was 90 min. The optimal resting tension was set at 20 mN in the first 30 min by gradually stretching the artery rings as in our previous work  and 3- [5- (tert-Butyl) isoxazol- 3- yl]- 2- [2- (3- chlorophenyl)hydrazono]- 3- oxopropanenitrile (ESI-09) was from Biolog Life Science Institute. PKI (14–22) amide (myristoylated) was obtained from Enzo Life Sciences. Brefeldin A was from LC laboratories and farnesyl thiosalicylic acid was from Cayman Chemical. All other chemicals were purchased from Sigma-Aldrich Corporation.The pretreatment of artery rings with the ARF-GEF inhibitor brefeldin A , a proven Epac inhibitor, significantly attenuated G-1-induced porcine coronary artery relaxation, the reduced relaxation was 44.90% of the total relaxation effect of G-1 , Table 1Measurement of Rap1 activity in porcine CASMCs  revealedVasodilator-stimulated phosphoprotein (VASP) can be phosphorylated by PKA at Ser 157, thus reflecting PKA activity . G-1 1 . AdditioPreviously we have shown that G-1 inhibits RhoA activity with a significant contribution of PKA to this inhibition . In the Increased RhoA activity leads to phosphorylation of MYPT1 at Thr-853 via Rho kinase , 25, whiIn another set of experiments, both Epac and PKA antagonists were used in examining the role of Epac as well as PKA in the inhibitory effect of G-1 on the phosphorylation of MYPT1 and MLC. When added separately, ESI-09 (10 μM) as well as PKI (5 μM) partially reversed the inhibitory effect of G-1 on phosphorylation of MYPT1 and MLC; however, when both of ESI-09 (10 μM) and PKI (5 μM) were present, the G-1 -induced inhibition of the phosphorylation of MYPT1 and MLC was completely reversed . These rThere is a growing interest in the role of GPER in cardiovascular regulation. The current work is the first to define the cAMP/ Epac signaling pathway in GPER-mediated coronary artery relaxation, which is mediated through attenuation of phosphorylation of MLCP and MLC via Rap1 inhibition of RhoA activity in parallel with PKA.GPER activation induces relaxation in a variety of vascular beds , 26, 27 The elevation in intracellular concentration of cAMP following activation of Gs-protein-coupled receptors is sufficient to activate Epac , a novel+ channel activity [Epac is the best described guanine nucleotide exchange factor (GEF) in the cardiovascular system and facilitates Rap1 cycling from the (GDP-bound) inactive state to the (GTP- bound) active state . Rap1 isactivity , 28.2+ level [2 analog, cicaprost, increased Rap1 activity and decreased RhoA activity [Increase in RhoA activity stimulated by various vasoconstrictors leads to Rho kinase-mediated phosphorylation of MYPT1, at Thr-853, not at Thr-696 , 25. The2+ level , 23, 35.2+ level , we studactivity . Activatactivity .In vascular smooth muscle cells, both PKA and Epac pathways are necessary for cell growth inhibition. Epac synergizes with PKA to mediate cAMP-induced cell growth arrest . In cAMPIn conclusion, the results of this study demonstrate that GPER-mediated porcine coronary relaxation involves both Epac and PKA signaling to inhibit the RhoA/ROCK effect on MLCP, and thereby, decrease phosphorylation of MLC and relaxation of coronary artery . These f


Several processes have been hypothesized to explain the slight overall expansion of Antarctic sea ice over the satellite observation era, including externally forced changes in local winds or in the Southern Ocean’s hydrological cycle, as well as internal climate variability. Here, we show the critical influence of an ocean–sea-ice feedback. Once initiated by an external perturbation, it may be sufficient to sustain the observed sea-ice expansion in the Ross Sea, the region with the largest and most significant expansion. We quantify the heat trapped at the base of the ocean mixed layer and demonstrate that it is of the same order of magnitude as the latent heat storage due to the long-term changes in sea-ice volume. The evidence thus suggests that the recent ice coverage increase in the Ross Sea could have been achieved through a reorganization of energy within the near-surface ice-ocean system. The mechanisms responsible for the overall expansion of Antarctic sea-ice in recent decades remain unclear. Here, using observations and model results, the authors show that ice-ocean feedbacks, triggered by an external perturbation, could be responsible for changes in sea-ice extent observed in the Ross Sea. This ultimately favors ice production and inhibits ice melt. The large upward oceanic heat flux to the ice in the Southern Ocean is indeed a central element in the sea-ice development and small variations of its magnitude have a large impact on the ice thickness and extent14. The role of changes in stratification is supported by several studies, showing in particular a surface freshening of the Ross Sea15 and a circumpolar intensification of near-surface vertical salinity and temperature gradients16.The overall positive trend in ice extent in the Southern Ocean since the late 1970s results from a strong increase in ice concentration  in the Ross and Weddell Seas, partly compensated by a decrease in the Bellingshausen Sea1 Fig. . This pa19. In particular, changes in ocean stratification and vertical oceanic heat flux are not necessarily caused by an external perturbation in freshwater forcing. Internal ice-ocean processes can also lead to a redistribution of the salt content, horizontally or vertically, producing potentially large changes in stratification on decadal to multi-decadal time scales. In particular, a mechanism related to the interplay between interannual variations and the seasonal cycle can engender a net vertical transport of salt in response to an initial perturbation, altering the near-surface stratification and amplifying initial changes20. Specifically, the mixed layer depth (MLD) under sea ice has a marked seasonality, with large values in winter associated with convection when sea ice forms and releases brine, and small ones in summer when sea ice melts and freshwater stabilizes the upper ocean column. The salt rejected during ice production in winter is thus mixed over a deep layer, while the freshwater due to melting is incorporated into a shallow mixed layer. In an equilibrium climate, if we neglect for simplicity the potentially important contribution of horizontal transport, the situation is stable because the same amount of salt transported to depth one year is entrained into the mixed layer the following year. However, if ice formation is particularly large during one or a few years, the brine released can be transported downward to deeper layers and not incorporated back into the mixed layer of the subsequent winters. This leads to a decrease in surface salinity, a stronger stratification, a shallower mixed layer and thus reduced vertical oceanic heat fluxes. Consequently, heat is trapped at depth in response to the larger sea-ice formation, leading to a positive feedback henceforth referred to as ice-coverage–ocean-heat-storage feedback.Internal climate system variability may have played a strong role in the observed sea-ice trendsHereunder, we show the signature of this feedback and quantify the heat trapped at the base of the ocean mixed layer in the Ross Sea, where long-term positive sea-ice trends are observed and simulated by a model. We demonstrate that the heat gain is of the same order of magnitude as the latent heat storage due to the associated changes in sea-ice volume, indicating that the trends may be sustained via a vertical redistribution of energy in the near-surface ice-ocean system. Conversely, this implies that enough energy is available at depth to melt the extra ice formed during the last few decades, should the mechanism be reversed.3 km2 yr−1) of the overall sea-ice extent increase around Antarctica (17.5 ± 4.1 103 km2 yr−1) over the last three decades1. Here, we study the changes in Antarctic sea-ice coverage using both the OSISAF21 sea-ice concentration satellite retrievals over 1979–2014 and sea-ice fields from a simulation performed with the global ocean–sea-ice model NEMO-LIM3.623, driven by atmospheric reanalyses . Except for the outer Weddell and inner Amundsen Seas, this simulation reproduces reasonably well the observed sea-ice concentration trends , and on ocean outputs from the model simulation. Compared to in situ observations, model results have the advantage of providing a complete spatial and temporal coverage. We evaluate the spatial correlation between trends in ice concentration and trends in ocean temperature at various depths  the sum of the latent heat changes associated with the sea-ice volume trends and the cooling at the surface over the concomitant heat content changes at depth. As illustrated in Fig. RH. On average over the Ross Sea in our 1979–2013 model simulation, RH approximately equals 1. Although the value of this coefficient is sensitive to the way the cooling and warming layers are defined (ranging from 0.5 to 3 for extreme definitions of those layers), this indicates that the heat losses in the ocean surface–sea-ice system and the heat gain at depth have the same order of magnitude. Unfortunately, the equivalent of RH cannot be evaluated from observations because of the lack of ice thickness measurements. Additional temperature measurements below the ice pack, especially in the poorly sampled inner Ross Sea, would also help to better evaluate RH and to improve estimates of the correlation with ice concentration. It is also possible to compute an equivalent to RH for the classical negative feedback14 in which an initial ice growth is partly compensated by a larger ocean-ice heat flux because of a mixed layer deepening and entrainment of warmer water. Nevertheless, as the time scale and processes are different for the decadal ice-coverage–ocean-heat-storage feedback discussed here, it may not be straightforward to make a connection between them.In order to quantify the impact of the ice-coverage–ocean-heat-storage feedback on the heat balance of the Ross Sea in the model, we have computed the ratio 31. Furthermore, the freshwater flux from icebergs and ice shelf melting is constant over the whole simulation. The enhancement of upper ocean stratification in Ross Sea regions associated with large ice concentration increases can thus not be due to a trend in precipitation or continental melt water input.The mechanism described above seems to explain well the observed and simulated changes in the Ross Sea. Observations are too scarce to unequivocally identify the causes of changes in MLD, but it is possible to test, in model results at least, if alternative mechanisms are also at play. Our simulation is performed using climatological run-off and precipitation forcing fields that were detrended32. This seems unlikely, first because a robust correlation of temperature trends at depth with ice concentration trends would not be expected if those two signals were, in fact, independent. Furthermore, Fig. A second hypothesis is that the subsurface temperature increase is primarily due to an independent warming and shoaling of CDW13. However, if winds and freshwater transport most probably play a role in initiating and amplifying the mechanism, our experiments show that their trends are not the primary cause of the simulated decadal sea-ice trends. The main evidence is provided in Fig. A third hypothesis is that the trends are simply forced by wind trends, which would induce a higher northward advection of sea ice and then freshwater. The important role of winds and sea-ice transport in many recent changes has been demonstrated in several studies33 in winter. Virtually everywhere else, areas in red illustrate the positive net balance in freshwater at the ocean surface, ensuing from the summer melt of ice advected away from the coast. Figure Advection still contributes to the trend and its effect is likely to be most prominent close to the ice edge. Trends are indeed large there in our results, suggesting a contribution of wind-induced changes, but positive ice concentration trends are also associated with a warming at depth in other regions such as the eastern inner Ross Sea. More generally, the trends in freshwater flux at the ocean surface resulting from sea-ice formation and melting Fig.  show no 13), the shallower mixed layer results in a stronger dilution of the mixed layer salt and thereby in a stronger surface salinity drop. Even without any further reductions in MLD, nonetheless expected, the shallower mixed layer can then lead to a steady surface freshening and stratification increase. In our simulation, Ross Sea trends in ice concentration do not correlate well in space with trends in surface freshwater input  whether or not models can represent the positive ice-coverage–ocean-heat-storage feedback, (2) the depth of its ocean temperature signature, and (3) its intensity through the heat storage coefficient RH we suggest here.Hence, vertical correlation profiles between ice concentration and ocean temperature trends Fig. , combineThe results indicate that the reorganization of heat within the upper-ocean–sea-ice column is by itself sufficient to sustain the recent sea-ice expansion in the Ross Sea, which accounts for most of the circumpolar expansion. The model analysis suggests that the proposed feedback may be amplified by wind trends and by a dilution effect related to freshwater export into regions experiencing mixed layer shoaling, but it may also occur without them. Our results further imply that the heat trapped in the warming layer of the Ross Sea represents enough energy to melt the ice formed previously, should the process be reversed and the heat released at the surface. Finally, the positive feedback described here may reconcile the various hypotheses existing to explain the recent Antarctic sea-ice trends: externally forced changes in ice dynamics (through changing wind regimes) or ocean freshwater balance (owing to increased precipitation or ice-sheet melting) may have provided the initial perturbation necessary to trigger an internal mechanism in the ocean–sea-ice system that sustains further sea-ice changes.27, and an estimate of the seasonal cycle  of temperature (T), salinity (S) and MLD. Specifically, this BOA-Argo-based climatology provides coefficients for the first two harmonics of seasonal variations, allowing computation of climatological values for any day of year. We first preselect temperature and salinity profiles with a minimum 25 m resolution in the top 350 m of the ocean and interpolate them to ensure complete coverage over this same depth range. Then, for each temperature or salinity record, a vertical profile of anomalies is obtained by subtracting the climatological estimate at the day of year and location of the observation. In order to obtain MLD from T and S profiles, preselected T and S profiles are paired using a criteria based on distance (<5 km), time (<25 h), and station number correspondence. From each T–S profile pair, we compute the associated MLD value following the T–S threshold criterion34 employed for the MLD climatology: the minimum of the depths z1, z2 satisfying | T(z1) − T(10m) | = 0.2, | S(z2) − S(10m) | = 0.03. An MLD anomaly is again obtained by subtracting the climatological estimate at the day of year and location of the observation.The analyses are based on the ocean temperature and salinity profiles from the BLUELink Ocean ArchiveT–S anomalies and MLD anomalies are then averaged annually on each cell of a regular 1° × 1° grid, extending from 40 to 80°S, using all records available within the corresponding grid cell every year. Linear regressions (over 1979–2014) can then be applied to the various fields independently. As the focus of our study is on multi-decadal time scales, only grid cells including records spread over at least 25 years were considered in order to compute trends.Working with anomalies is critical here to sidestep the important issue of seasonal biases in the sampling of the ocean. Profiles of 21 Global Sea Ice Concentration Climate Data Records (1978–2015). These data were interpolated on the same grid as the one for ocean observations, and long-term trends in yearly-averaged ice concentration were computed in each grid cell.We use sea-ice concentration satellite data from the OSISAFAll observation-based trends are computed over 1979–2014. Indeed, the reprocessed data set we use for ice concentration covers up to April 2015 only. After this date, another near real-time OSISAF product exists (continuous reprocessing offline product), but it is not fully consistent with the reprocessed data set, which makes it unsuitable for our application.23. The standard simulation analyzed here was performed over 1958–2013 using DFS5.2  atmospheric forcing fields31. An additional sensitivity experiment was conducted in order to assess the role of winds in the presented mechanism  was performed to study the changes over the period 1952–1978  are also shown in gray.In order to produce the correlations displayed in Fig. RH, used to compare the amount of energy stored in subsurface layer to the heat losses associated with sea-ice volume changes and the ocean surface cooling, was defined as:ρice = 917 kg m−3, L = 334 × 103 J kg−1, ρsw = 1025 kg m−3, and csw = 4 × 103 J kg−1 K−1 are the selected values for sea-ice density, the pure ice latent heat of fusion, the seawater density, and specific heat capacity, respectively. Δvice represents the ice volume change per unit area over 1979–2013  in the considered grid cell, ΔTcl and ΔTwl are the temperature changes in the cooling and warming layers over the same period, and dzcl and dzwl are the thicknesses of the associated layers (Fig. RH was first computed in each model grid cell and was finally spatially averaged over the Ross Sea sector.The heat storage ratio entclass1pt{minimahttps://researchdata.ands.org.au/bluelink-ocean-archive/692138?source=suggested_datasets, and the OSISAF sea-ice concentration satellite product at http://osisaf.met.no. The MATLAB code produced in order to perform all the data processing and analyses of this study is available upon request to the authors.All observational data supporting the findings of this study are publicly available online. BOA hydrographic data are available at Supplementary InformationPeer Review File


Autumn sea ice trends in the western Ross Sea dominate increases in Antarctic sea ice and are outside the range simulated by climate models. Here we use a number of independent data sets to show that variability in western Ross Sea autumn ice conditions is largely driven by springtime zonal winds in the high latitude South Pacific, with a lead-time of 5 months. Enhanced zonal winds dynamically thin the ice, allowing an earlier melt out, enhanced solar absorption, and reduced ice cover the next autumn. This seasonal lag relationship has implications for sea ice prediction. Given a weakening trend in springtime zonal winds, this lagged relationship can also explain an important fraction of the observed sea ice increase. An analysis of climate models indicates that they simulate weaker relationships and wind trends than observed. This contributes to weak western Ross Sea ice trends in climate model simulations. Antarctic sea ice extent continues to increase, with autumn sea ice advances in the western Ross Sea particularly anomalous. Here, based on analysis of independent datasets, the authors show that springtime zonal winds in the high latitude South Pacific drive western Ross Sea autumn sea ice conditions. The observed trend is made up of large regional changes that are partly compensating. While previous work has indicated that the observed increases in the total Antarctic sea ice extent are consistent with natural variability simulated by climate models4, this is not the case if the regional and seasonal nature of the trends is considered.Observed Southern Hemisphere sea ice extent has increased since 19795. Additionally, the western Ross Sea ice trends do not appear attributable to observed changes in many modes of variability6, including the significant trends in the austral summer Southern Annular Mode7, which are associated with stratospheric ozone loss. A number of previous studies11 have identified mechanisms that can cause increased sea ice around Antarctica. However, these generally do not explain the increases observed in the western Ross Sea during autumn.Of particular interest is the western Ross Sea region during the autumn ice advance season. This is the region and season of the largest ice increases in the Southern Hemisphere Fig. . These o12. Observational evidence has also indicated that seasonal-lagged relationships influence ice trends. In particular, ice concentration trends in autumn have been related to changes in ice during the previous spring season13. The presence of seasonal memory in Antarctic sea ice-ocean interactions has also been documented in the context of interannual variability16, with ice conditions during the autumn ice advance season significantly related to sea ice during the previous spring ice retreat.Trends in the Ross Sea ice extent have been associated with coincident wind-driven ice transport anomaliesHere we use a number of independent data sets to assess factors that contribute to variability and change in western Ross Sea ice cover during autumn. This includes the analysis of mechanisms driving a seasonal memory of the sea ice and the possible consequences for observed sea ice trends in the western Ross Sea. We show that, on interannual timescales, wind variability in the previous October is a strong predictor of ice anomalies in March to May, with implications for seasonal ice predictability. On the basis of these seasonal-lagged relationships, we find that trends in springtime winds could account for up to 30% of the autumn sea ice trends in the western Ross Sea. Climate models simulate weaker relationships and wind trends than observed, which influences the magnitude of western Ross Sea ice trends that they simulate.R = 0.85, 0.74, 0.65 for AMJ, respectively). This suggests that much of the ice variability in autumn in the western Ross Sea is associated with variations in the ice advance beginning in March.We assess 1979–2015 autumn conditions in the western Ross Sea, defined as the region between 150 and 200º E, as this is the area with the largest trends in an absolute sense and also relative to the interannual variability Fig. . The lon12. Correlation analysis using the ERA-Interim data indeed suggests that the western Ross Sea March ice area anomalies are strongly related to wind variability. However, somewhat counter to expectations, the ice area anomalies only show minimal correlation to the simultaneous March or prior February winds  with variability in the depth of the October Amundsen Sea Low (ASL), which is a climatological low pressure center in the region18. Many modes of variability such as the El Niño-Southern Oscillation19 (and associated Pacific–South American pattern), the Southern Annular Mode19, the Pacific Decadal Oscillation11 and the Atlantic Multi-decadal Oscillation20, influence the ASL, providing links to tropical and global variability. The correlation of detrended western Ross Sea ice and the October ASL is R = 0.50, meaning that a deeper October ASL is associated with less March ice cover. This is compared to a correlation of R = −0.75 for the zonal wind index. Interestingly, the relationship between March sea ice area in the western Ross Sea and the depth of the ASL is of opposite sign for the seasonal-lagged relationships discussed here compared to a contemporaneous relationship21. The fact that the zonal wind shows stronger relationships to western Ross Sea ice area than the ASL pressure alone suggests that various factors such as the location of the ASL also may play a role. This is generally consistent with other work assessing concurrent relationships that has indicated that not only the depth of the ASL but also its location can affect relationships to sea ice22.Sea ice area correlations are stronger with the zonal wind than with other atmospheric circulation metrics  response can differ markedly from the longer term  response due to the influence of ocean circulation changes. Nevertheless, if we make these assumptions , we find that the trends in the zonal winds in October can account for 20–30% of the trend in the western Ross Sea ice area in March and April 32, which is available from 1982 to 2015.We assess the relationship of wind variations to a number of sea ice conditions, including ice concentration, ice motion, and the timing of ice retreat and advance for 1979–2015. This makes use of the SMMR/SSMI Bootstrap sea ice concentration33. Twenty different climate models are considered as shown in Supplementary Table Climate model analysis uses output from pre-industrial control simulations that have been included in the Coupled Model Intercomparison Project version 5 (CMIP5)Western Ross Sea ice area is defined as the ice area between 150 and 200º E.http://apps.ecmwf.int/datasets/. The sea ice data are available from the National Snow and Ice Data Center31 (http://nsidc.org/). NOAA’s OISSTv2 is available from https://www.esrl.noaa.gov/psd/data/gridded/data.noaa.oisst.v2.highres.html. Data from CMIP5 models is available from http://cmip-pcmdi.llnl.gov/cmip5/. Information on various data sets was obtained from the Climate Data Guide34.The data that support the findings from this study are available in a number of public repositories. ERA-Interim data are available from Supplementary InformationPeer Review File