Please Register or Sign in to view this content Quantum Commodity Intelligence is a premium paid subscription service for professionals in the oil Quantum Hydrogen service subscribers have access to: Get in touch with us for subscription information on all Quantum platforms The Heinsberg district police authority and the police in Hückelhoven are looking forward to the construction of a new modern police station in the town of Hückelhoven The investor and landlord for this project will be the ARGE Rathausquartier Hückelhoven consortium consisting of the city of Hückelhoven and Stadtentwicklung Hückelhoven GmbH & Co The rental agreement for the new police station was signed today The General Representative of the District Administrator and the First Alderman of the City of Hückelhoven have thus paved the way for this forward-looking construction project The new police station will be built on Martin-Luther-Straße in the north of the city and will comprise a rental area of around 1800 m² the 1st and 2nd floors and parts of the 3rd floor It will house the security and district services as well as the criminal investigation department the detailed construction planning phase will now begin Construction is scheduled to begin in mid-2026 and the new police station is due to be completed and handed over in summer 2028 This modern facility will meet current standards and offer police officers a contemporary working environment in the heart of Hückelhoven The current police station building is outdated and no longer meets today's requirements The employees of the Hückelhoven police are therefore looking forward with great anticipation to the move to the new premises which will not only be functional but also architecturally appealing is satisfied that the search for a new location for the police station in Hückelhoven has now led to a good result "We are delighted that the new police station will give us a presence in the heart of Hückelhoven and make us even more accessible to the people." "Having a police station in the center of the city is certainly a major concern of every mayor It also fulfills the wish of the council and administration to have the new police station as an essential building block for the new quarter at the town hall." This new police station represents significant progress for the safety and urban development of Hückelhoven and will have a positive impact on the entire region Metrics details Third-generation chimeric antigen receptor T cells (CARTs) for relapsed or refractory (r/r) chronic lymphocytic leukemia (CLL) may improve efficacy compared to second-generation CARTs due to their enhanced CAR design We performed the first phase 1/2 investigator-initiated trial evaluating escalating doses of third-generation CARTs (HD-CAR-1) targeting CD19 in patients with r/r CLL and B-cell lymphoma CLL eligibility criteria were failure to two therapy lines including at least one pathway inhibitor and/or allogeneic hematopoietic cell transplantation Nine heavily pretreated patients received HD-CAR-1 at dose levels ranging from 1 × 106 to 200 × 106 CART/m2 In-house HD-CAR-1 manufacturing was successful for all patients one case of grade 3 cytokine release syndrome was observed HD-CAR-1 products of responders contained significantly more CD4 + T cells compared to non-responders a strong enrichment of effector memory-like CD8 + T cells with high expression of CD39 and/or CD197 was observed HD-CAR-1 demonstrated encouraging efficacy and exceptionally low treatment-specific toxicity presenting new treatment options for patients with r/r CLL liso-cel has recently been approved by the U.S Food and Drug Administration as the first second-generation CART product for adults with relapsed or refractory (r/r) CLL or small lymphocytic lymphoma RV-SFG.CD19.CD28.4-1BBzeta retroviral vector supernatant was provided by Prof Malcolm Brenner from Baylor College of Medicine in Houston This CAR harbors the costimulatory domains CD28 and 4-1BB The trial was approved by the institutional review board as well as by the German federal regulatory authority for immunotherapy (Paul-Ehrlich-Institut and written informed consent was obtained from all participants The trial was conducted in compliance with the principles of the Declaration of Helsinki Samples were measured on Cytek Aurora flow cytometer (Cytek Biosciences responders were defined as patients who achieved a CR after CART infusion that was sustained at least for 2 months All non-responders had undergone prior alloHCT Significance levels were determined by a two-sided Welch’s t-test CD4+ and CD8 + T cells were separated and clustered independently using the same approach as described above Differential abundance of cells within the respective UMAP space was highlighted by density plots For quantification of differentially abundant CD4+ or CD8 + T-cell subtypes between responders and non-responders the frequency of each T-cell subtype was calculated per sample the frequency of responders was divided by the corresponding mean frequency of non-responders These responder-specific fold-changes were log2 transformed and visualized in boxplots Statistics were calculated using Prism Software (version 10.2.0; Graphpad Software Inc. Progression-free survival (PFS) was determined measuring the duration from the date of CART administration until clinical progression Survival curves were compared using log-rank testing A p-value less than 0.05 was considered statistically significant Eight patients with r/r CLL were enrolled between October 2018 and May 2023. As the trial cohort was already fully recruited, another patient with r/r CLL was treated compliant with the trial but formally off-study in July 2023 after having given fully informed consent (#9). Baseline characteristics of all nine patients are listed in Table 1 Patients had a median age of 60 years (range 45 to 68) and had received a median of 5 (range 2 to 10) prior treatment lines Four patients (44%) had received prior alloHCT Seven patients (78%) harbored TP53 abnormalities All patients had failed Bruton’s tyrosine kinase inhibitors (BTKi) and all had received at least one venetoclax-based regimen Eight of the nine patients were refractory to venetoclax-based treatment as well bridging therapy was administered to all patients mostly with venetoclax-antibody combinations resulting in CR and partial remission (PR) in three and two patients all patients had flow-detectable MRD at lymphodepletion One patient (#8) had a history of Richter transformation which however was not present at the time of enrollment All patients received at least one dose of HD-CAR-1 CARTs (Table 2) Dose levels (DL) were DL1 (1 × 106 CARTs/m2) in one patient DL5 (10 × 107 CARTs/m2) in two patients and DL6 (20 × 107 CARTs/m2) in five patients HD-CAR-1 CARTs were well tolerated (Table 2) Although seven of nine patients experienced CRS higher grade CRS was observed only in a single patient (11%) Early ICAHT occurred in eight patients (89%) Late ICAHT was observed as grade 1 in one patient (#9) and as grade 2 in two patients (#2 and #7) without the need for granulocyte colony-stimulating factor support Hypogammaglobulinemia was observed in eight of nine patients at lymphodepletion One of these patients showed recovery of gamma globulin levels after HD-CAR-1 treatment (#5) One patient (#2) did not exhibit hypogammaglobulinemia at lymphodepletion or after day 90 post CART infusion Intravenous immunoglobulins were administered to three patients following HD-CAR-1 treatment (#1 The only early infections post-CART infusion occurred in patient #5 who presented herpes simplex virus type 1 infection of the lower lip and respiratory infection without pathogen identification. Late infections (beyond day 30) occurred in four patients (#1-3 and #7; Table 2) Median duration of response (DOR) was 6.4 months A Progression-free survival (PFS) of all patients achieving a complete remission (CR) after CART administration vs B Overall survival (OS) and PFS of all patients after CART treatment C Swimmer plots of all patients after CART administration treated at dose level 5 or higher : CART therapy; : progressive disease; : stable disease; : partial remission; : MRD-positive CR; : MRD-negative CR; : death; : 2nd allogeneic hematopoietic cell transplantation; : radiation therapy; A acalabrutinib A HD-CAR-1 CART products of seven CLL patients (#1-7) were analyzed with spectral flow cytometry using a 36-marker panel and computational analysis (see methods) B The downsampled subset of cells from all seven CART products is presented with uniform manifold approximation and projection (UMAP) visualization Clusters are labeled depending on surface marker expression and displayed in different colors C Frequencies of CD4+ (left) and CD8+ (right) T cells within the CART products of responders (R) vs non-responders (NR) are displayed as bloxplots CD8+ D and CD4+ G T-cell subsets extracted from all seven CART products are presented with UMAP visualization and clusters are annotated based on surface marker expression and displayed in different colors Differential compositions between R and NR of CD8+ E and CD4+ H T-cell subsets are displayed as density plots Differential frequencies between R and NR of specific CD8+ F and CD4+ I T-cell subtypes are presented as boxplots of log2 fold-changes Higher abundance in R is indicated as positive log2 fold change and negative log2 fold changes visualize higher frequencies in NR While the small sample size is an obvious limitation of this study a major strength is its prospective design demonstrating an extremely favorable safety profile and high response rate of the HD-CAR-1 approach tested here in heavily pretreated/double-refractory patients with CLL and potentially broadening patient eligibility for CART therapy in CLL treatment of heavily pretreated patients with high-risk CLL having failed multiple pathway inhibitors with the third-generation CART HD-CAR-1 is feasible and associated with only very modest CART-specific toxicity The preliminary efficacy signals obtained suggest that HD-CAR-1 can induce prolonged complete responses in otherwise refractory patients and warrant further exploration of this approach Original data are available with Patrick.Derigs@med.uni-heidelberg.de upon request KTE-X19 CAR T-cell therapy in relapsed or refractory mantle-cell lymphoma KTE-X19 for relapsed or refractory adult B-cell acute lymphoblastic leukaemia: phase 2 results of the single-arm Tisagenlecleucel in adult relapsed or refractory diffuse large B-cell lymphoma Axicabtagene ciloleucel as first-line therapy in high-risk large B-cell lymphoma: the phase 2 ZUMA-12 trial Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia Axicabtagene ciloleucel as second-line therapy for large B-cell lymphoma Tisagenlecleucel in adult relapsed or refractory follicular lymphoma: the phase 2 ELARA trial Lisocabtagene maraleucel versus standard of care with salvage chemotherapy followed by autologous stem cell transplantation as second-line treatment in patients with relapsed or refractory large B-cell lymphoma (TRANSFORM): results from an interim analysis of an open-label Lisocabtagene maraleucel for patients with relapsed or refractory large B-cell lymphomas (TRANSCEND NHL 001): a multicentre seamless design study CAR-modified cellular therapies in chronic lymphocytic leukemia: is the uphill road getting less steep Axicabtagene ciloleucel in relapsed or refractory indolent non-Hodgkin lymphoma (ZUMA-5): a single-arm Three-year follow-up analysis of axicabtagene ciloleucel in relapsed/refractory indolent non-Hodgkin lymphoma (ZUMA-5) Durable response after tisagenlecleucel in adults with relapsed/refractory follicular lymphoma: ELARA trial update T cells from CLL patients exhibit features of T-cell exhaustion but retain capacity for cytokine production Immune dysfunction in chronic lymphocytic leukemia: the role for immunotherapy Chronic lymphocytic leukemia T cells show impaired immunological synapse formation that can be reversed with an immunomodulating drug Perturbation of the normal immune system in patients with CLL Immune dysfunctions and immune-based therapeutic interventions in chronic lymphocytic leukemia Control of chronic lymphocytic leukemia development by clonally-expanded CD8(+) T-cells that undergo functional exhaustion in secondary lymphoid tissues Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia Redirecting T cells to glypican-3 with 4–1BB zeta chimeric antigen receptors results in Th1 polarization and potent antitumor activity 4–1BB and CD28 signaling plays a synergistic role in redirecting umbilical cord blood T cells against B-cell malignancies Chimeric antigen receptors combining 4–1BB and CD28 signaling domains augment PI3kinase/AKT/Bcl-XL activation and CD8+ T cell-mediated tumor eradication Micromolar affinity CAR T cells to ICAM-1 achieves rapid tumor elimination while avoiding systemic toxicity established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains A phase I/IIa trial using CD19-targeted third-generation CAR T cells for lymphoma and leukemia Treatment of patients with relapsed or refractory CD19+ lymphoid disease with T lymphocytes transduced by RV-SFG.CD19.CD28.4-1BBzeta retroviral vector: a unicentre phase I/II clinical trial protocol Improvement of in vitro potency assays by a resting step for clinical-grade chimeric antigen receptor engineered T cells Treatment of adult ALL patients with third-generation CD19-directed CAR T cells: results of a pivotal trial Immune effector cell-associated hematotoxicity: EHA/EBMT consensus grading and best practice recommendations ASTCT consensus grading for cytokine release syndrome and neurologic toxicity associated with immune effector cells Side-effect management of chimeric antigen receptor (CAR) T-cell therapy Improving efficiency and sensitivity: European Research Initiative in CLL (ERIC) update on the international harmonised approach for flow cytometric residual disease monitoring in CLL Optimized assessment of qPCR-based vector copy numbers as a safety parameter for GMP-grade CAR T cells and monitoring of frequency in patients Healy J UMAP: Uniform manifold approximation and projection for dimension reduction Ripley BD Modern Applied Statistics with S multimodal and scalable single-cell analysis B and NK lymphocyte subpopulations in peripheral blood of healthy Cuban adults Chronic lymphocytic leukemia: 2022 update on diagnostic and therapeutic procedures Genomics of resistance to targeted therapies Single-cell multiomics reveal the scale of multilayered adaptations enabling CLL relapse during venetoclax therapy Mechanisms of resistance to noncovalent Bruton’s tyrosine kinase inhibitors High-risk chronic lymphocytic leukemia in the era of pathway inhibitors: integrating molecular and cellular therapies Feasibility and efficacy of CD19-targeted CAR T cells with concurrent ibrutinib for CLL after ibrutinib failure Durable molecular remissions in chronic lymphocytic leukemia treated with CD19-specific chimeric antigen receptor-modified t cells after failure of ibrutinib B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells Chemotherapy- refractory diffuse large B-cell lymphoma and indolent B-cell malignan- cies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia Long-term outcomes from a ran- domized dose optimization study of chimeric antigen receptor mod- ified T cells in relapsed chronic lymphocytic leukemia an Anti-CD19 Chimeric Antigen Receptor (CAR) t-cell therapy in patients with relapsed/refractory chronic lymphocytic leukemia Lisocabtagene maraleucel in chronic lymphocytic leukaemia and small lymphocytic lymphoma (TRANSCEND CLL 004): a multicentre Rituximab maintenance versus observation alone in patients with chronic lymphocytic leukaemia who respond to first-line or second-line rituximab-containing chemoimmunotherapy: final results of the AGMT CLL-8a Mabtenance randomised trial Cytotoxicity of the CD3×CD20 bispecific antibody epcoritamab in CLL is increased by concurrent BTK or BCL-2 targeting Long-term outcomes from a randomized dose optimization study of chimeric antigen receptor modified t cells in relapsed chronic lymphocytic leukemia Third-Generation Chimeric Antigen Receptor (CAR) T Cells in Patients with Relapsed/Refractory Acute Lymphoblastic Leukemia (ALL) and Non-Hodgkin Lymphoma (NHL) - Results from the Heidelberg Trial 1 (HD-CAR-1 trial) Feasibility and safety of CD19 chimeric antigen receptor T cell treatment for B cell lymphoma relapse after allogeneic hematopoietic stem cell transplantation Decade-long leukaemia remissions with persistence of CD4 4-1BB and optimized CD28 co-stimulation enhances function of human mono-specific and bi-specific third-generation CAR T cells Blinatumomab nonresponse and high-disease burden are associated with inferior outcomes after CD19-CAR for B-ALL Download references This work was supported by the National Center for Tumor Diseases (NCT) by the German Cancer Research Center (DKFZ) by the Jochen Siebeneicher Foundation and by a personal donation from Dr PDe is supported by the Clinician Scientist Fellowship Program of the DKFZ (supported by the Dieter Morszeck Foundation) Open Access funding enabled and organized by Projekt DEAL Carsten Müller-Tidow & Michael Schmitt Berlin Institute of Health (BIH) at Charité – Universitätsmedizin Berlin Berlin Institute for Medical Systems Biology Max Delbrück Center for Molecular Medicine in the Helmholtz Association German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ)/National Center for Tumor Diseases (NCT) Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM gGmbH) Precision Healthcare University Research Institute All authors revised the manuscript and approved the final version of the manuscript All authors are accountable for all aspects of the work AS: Travel grants from Hexal and Jazz Pharmaceuticals Co‐founder and part‐time employee of TolerogenixX LtD Grants and/or provision of investigational medicinal products from Pfizer Financial support for educational activities and conferences from bluebird bio Co‐Founder and shareholder of TolerogenixX Ltd Roche; research support from Neovii and Riemser None of the mentioned sources supported the work described within this manuscript All methods were performed in accordance with the relevant guidelines and regulations The HD-CAR-1 trial was approved by the institutional review board as well as by the German federal regulatory authority for immunotherapy (Paul-Ehrlich-Institut Written informed consent was obtained from all participants Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41375-024-02392-7 Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article You can read this article in 2 minutesNatalia Jakubowska Rhenus Warehousing Solutions has announced it is taking over the consolidation and deconsolidation for C&A while the Nagel Group is expanding its warehouse capacities The clothing supplier C&A is now using the expertise of Rhenus Warehousing Solutions at the Hückelhoven location to consolidate and deconsolidate its goods The location acts as a central hub for Europe and serves all European distribution centers The logistics centre has a hall area of ​​120,000 square metres and is located near the Dutch border and important transport hubs pharmaceutical & healthcare and retail & fashion sectors “We are very proud to welcome C&A as a new customer and look forward to a long-term partnership The commitment of an internationally operating company to handle its warehouse logistics via our location in Hückelhoven confirms the quality of our services and our infrastructure,” says Andreas Mayer member of the management team at Rhenus Warehousing Solutions Germany the Nagel-Group has put a new multi-temp system into operation in Schweitenkirchen The new cold storage halls offer space for 13,200 pallets The Nagel Group has invested 40 million euros in the further development of the system “The logistics center in Schweitenkirchen is of great importance for the south of Germany and the connection to Austria,” says Marion Nagel Chairman of the Board of Directors of the Nagel Group The new facility enables customers to offer logistics solutions in all temperature ranges from storage to transshipment and transport as well as value added services the Nagel Group also had the Expansion of their logistics location in Nuremberg completed The multi-temp system there is now equipped with an automated high-bay warehouse photovoltaic systems and storage capacities for all temperature ranges “The modern facilities in Schweitenkirchen and Nuremberg Chief Operating Officer at the Nagel-Group Pölös Zsófia Journalist Trans.info | 6.05.2025 Fragrance on the Fly: Why Pocket Perfumes Are Perfect for Airplane TravelSponsored Article 6.05.2025 Volume 9 - 2018 | https://doi.org/10.3389/fimmu.2018.02207 a severe complication of allogeneic hematopoietic stem cell transplantation significantly affects the post-transplant morbidity and mortality Systemic steroids remain the gold standard for the initial management of GvHD up to 60% of patients will not sufficiently respond to steroids has shown good clinical results in such steroid-refractory/resistant GvHD patients but not global immunosuppressive and steroid-sparing capacity the balance of immune cells is destroyed: effector cells are not any longer efficiently controlled by regulatory cells the precise mechanism and the impact of ECP on anti-viral/anti-leukemic function remain unclear 839 ECP treatments were performed on patients with acute GvHD (aGvHD) and chronic GvHD (cGvHD) A comprehensive analysis of effector and regulatory cells in patients under ECP therapy included multi-parametric flow cytometry and tetramer staining interferon-γ enzyme-linked immunospot Gene profiling of myeloid-derived suppressor cells (MDSCs) was performed by microarray analysis modulations of effector and regulatory cells as well as proinflammatory cytokines were observed under ECP treatment: (1) GvHD-relevant cell subsets like CD62L+ NK cells and newly defined CD19hiCD20hi B cells were modulated but (2) quantity and quality of anti-viral/anti-leukemic effector cells were preserved (3) The development of MDSCs was promoted and switched from an inactivated subset (CD33−CD11b+) to an activated subset (CD33+CD11b+) (4) The frequency of Foxp3+CD4+ regulatory T cells (Tregs) and CD24+CD38hi regulatory B cells was considerably increased in aGvHD patients and Foxp3+CD8+ Tregs in cGvHD patients (5) Proinflammatory cytokines like IL-1β and TNF-α were significantly reduced ECP constitutes an effective immunomodulatory therapy for patients with steroid-refractory/resistant GvHD without impairment of anti-viral/leukemia effects the efficacy of allo-HSCT strongly relies on the graft-vs.-leukemia (GvL) effect which is tightly linked to GvHD strategies for GvHD treatment that are efficient steroid-sparing and not compromising the beneficial anti-leukemia and anti-viral immunity are highly desirable Extracorporeal photopheresis (ECP) is a second-line treatment for GvHD, which has been associated with good clinical responses. It employs (i) apheresis with ex vivo collection of peripheral mononuclear cells, (ii) photoactivation with exposure of leukocyte-enriched plasma to the photosensitizing agent 8-methoxypsoralen and ultraviolet A light, (iii) reinfusion of such physico-chemically modified ECP-treated cells to the patient. In a pooled analysis (6) overall response rates (ORR) were 69% and 64% for acute and chronic GvHD the immunomodulation of other immune regulatory cells effector cells and proinflammatory cytokines influencing the success of the ECP treatment remains to be elucidated This study was performed to address these unsolved questions Twenty patients with steroid-refractory/resistant aGvHD ≥ II and moderate to severe cGvHD received ECP therapy at the University Hospitals Heidelberg and Greifswald in Germany. The diagnosis of steroid-refractory/resistant GvHD is based on the European recommendations (14, 15) Adequate venous access and leukocytes > 1/nl were required to be eligible for ECP The study was approved by the Institutional Review Board Each ECP treatment was administered over two consecutive days using the Therakos UVAR XTS photopheresis system. For patients with aGvHD, 12 weeks of intensive, semiweekly (twice per week) treatment, were followed by biweekly (every 2 weeks) ECP treatment (16, 17) Patients with cGvHD received either an 8-week intensive treatment followed by a biweekly treatment or a biweekly treatment upfront ECP therapy was stopped when patients either achieved complete response (CR) or maximal partial response (PR) with steroid reduction Blood was drawn from consenting patients from the first therapy and every second to fourth ECP cycle before the ECP treatment process PBMCs were diluted 2:1 with phosphate-buffered saline (PBS) then isolated by density gradient centrifugation (2,000 rpm without break) and stored in liquid nitrogen room temperature) and stored at −80°C After thawing, PBMCs were rested overnight as described earlier (18) followed by CD8 MicroBeads separation according to the manufacture's instruction (Miltenyi Biotec) CD56+ NK cells were enriched by negative selection with NK cell isolation kit according to the manufacturer's instructions (Miltenyi Biotec) MDSCs subsets were sorted by FACSAria (BD biosciences) using CD11b allophycocyanin (APC) (clone: ICRF44 CD33 fluorescein isothiocyanate (FITC) (clone: HIM3-4 HLA-DR Peridinin chlorophyll (PerCP) (clone: L243 cells were either stained with antibodies for 15–30 min at 4°C HLA-DR) or 37°C [C-C chemokine receptor type 7 (CCR7)] in the dark followed by washing once with FACS buffer (1% bovine serum albumin and 2 mM Ethylenediaminetetraacetic acid in PBS) 1 × 106 cells were incubated with cytomegalovirus (CMV) phosphoprotein65495−503 (pp65495−503) (CMV-A2)/HLA-A*0201 tetramer and CCR7 antibody for 15 min at 37°C followed by staining with other antibodies for 20 min at 4°C in the dark activated cells which were stimulated with 1 μg/ml staphylococcal enterotoxin B (SEB) (Sigma-Aldrich) in the presence of brefeldin A (Biolegend) for 6 h were fixed and permeabilized by using Miltenyi Forkhead box proteins3 (Foxp3) fix/perm buffer set then finally stained for 30 min with FoxP3 and IL-17a antibody 1 x 105 CD8+ cells were mixed with auto-CD8− cells at a ratio of 1:1 and plated in triplicate. CMV/Epstein-Barr virus/Influenza virus (CEF) Pool (extended) (JPT Peptide Technologies) was added directly to the experimental well at a concentration of 2 μg/ml/peptide. ELISpot assay was performed according to the manufacturer's instructions, as described previously (21) Image analysis of ELISpot plates was performed with an ImmunoSpotTM Analyzer (Cellular Technology Limited) A 4-h 51Cr release assay was performed to test the NK activity, as described previously (22) target cells K562 labeled with 51Cr were cocultured with effector CD56+ NK cells at effector-to-target cell ratios ranging from 50:1 to 6:1 Maximal release and spontaneous release were determined by incubating the target cells with 1% Triton X-100 (Sigma-Aldrich) and medium alone NK activity was calculated by the following formula: % specific lysis = [mean count per minute (c.p.m.) (experimental release)–mean c.p.m Total RNA was extracted with the RNeasy Micro kit (QIAGEN) from sort-purified MDSCs and gene expression determined using Affymetrix GeneChip®; Human Genome U133 Plus 2.0 Arrays mRNA was amplified and biotinylated with Affymetrix 3′ IVT Pico Reagent Kit before hybridization Gene Expression Microarrays were scanned using the Affymetrix GeneChip®; Scanner 3000 Quantification of the cytokines IL-1β and tumor necrosis factor-α (TNF-α) in human serum samples was conducted by the LUNARISTM Human 6-Plex Ophthalmology Kit384 (AYOXXA Biosystems) according to the manufacturers' instructions Results were analyzed using the LUNARISTM Analysis Suite Software Gene expression was assessed after adjustment by the Benjamini-Hochberg procedure Differences in cell frequency between before and post ECP therapy were assessed by paired-sample T test The significant difference between healthy donors (HDs) and GvHD patients was assessed by independent T test a p-value < 0.05 was considered to be statistically significant A total of 20 patients suffering from GvHD were treated by ECP. Nine aGvHD patients were treated with ECP in addition to immunosuppressive therapies including steroids, calcineurin inhibitors and/or mycophenolate mofetil (MMF) as well as pentostatin and/or ruxolitinib (Table 1). Eleven patients with cGvHD received ECP treatment despite triple drug therapy comprising of steroids, cyclosporine A (CsA), mTOR inhibitors, and/or MMF (Table 1) one patient with aGvHD (patients #1) and two patients with cGvHD (patients #15 and #18) had to be withdrawn after only four to five ECP cycles due to pancytopenia which were not associated with toxicity of ECP therapy All patients showed neither increased susceptibility to infections nor reactivation of CMV nor loss of complete chimerism during ECP therapy (Table 1) Differentiation and education of NK cell populations by ECP in aGvHD patients The assessment of CD56briCD16− NK cells (A) and CD56dimCD16+ NK cells (C) before and after ECP therapy shows that ECP treatment can promote the development of NK cells from CD56briCD16− NK cells to CD56dimCD16+ NK cells as well as educate NK cells by decreasing expression of NKG2D and CD62L (B,D) (A) shows representative dot plots of CD19hiCD20hi B cells among HD (B) displays the frequency of CD19hiCD20hi B cells in both aGvHD and cGvHD groups prior to ECP treatment (C) Characterization of CD19hiCD20hi B cells showed significantly lower expression of BAFF-R and CD38 but slightly increased CD24 expression (D,E) When compared to CD19+CD20+ B cells CD19hiCD20hi B cells showed a different component pattern a significantly higher BAFF-R+CD38− proportion and memory B cells Dashed lines represent the corresponding median value of frequencies observed in 25 HDs Differences in cell frequency between different groups were assessed by Independent T test the homogeneous CD14+HLA-DR−/low MDSC population with a normalization was observed (data not shown) CD14+HLA-DR−/low MDSC subpopulations in the peripheral blood of GvHD patients with ECP treatment The immunophenotype of MDSCs was assessed by flow cytometry transitional and activated subsets were observed within CD14+HLA-DR−/low MDSCs among aGvHD patients cGvHD patients and healthy donors (HDs) suggesting a development from inactivated into activated MDSCs (B) A reduction of inactivated MDSCs was observed after ECP therapy in aGvHD patients (C) The volcano plot shows the gene expression between activated MDSCs and inactivated MDSCs The horizontal axis represents the fold change in intensity and the vertical axis represents statistical significance (Log Odds) The bar chart indicates the differential gene expression between activated and inactivated MDSCs Differences in cell frequency between different groups were assessed by paired-sample T test our data indicated activated MDSCs have stronger immunosuppressive potency Immunomodulation of regulatory T and B cells through ECP and CD24+CD38hi Bregs (C) were monitored in patients with aGvHD and cGvHD before and after ECP therapy Foxp3+CD8+ Tregs significantly increased under ECP therapy in both aGvHD and cGvHD patients along with significant up-regulation of Foxp3+CD4+ Tregs and Bregs in aGvHD patients Elevated levels of proinflammatory cytokines (IL-1β, IL-6, and IL-8) were found even on high doses of steroids e.g., 2 mg/kg body weight. ECP had a positive effect in all cases with a decline of proinflammatory cytokines (Figure 5). High peaks of cytokines IL-1β (Figure 5A) and TNF-α (Figure 5D) in patient #7 caused by rapid steroid reduction were returned back to low levels by ongoing ECP treatment A fast reduction of proinflammatory cytokines IL-1β (A) and TNF-α (D) was observed in all patients Patient #7 showed a rebound of IL-1β and TNF-α after rapid steroid reduction Eventually the level of both cytokines decreased when ECP was continued Dashed lines represent the corresponding median value of cytokine levels observed in healthy donors The frequency of ECP cycles is indicated on the x-axis The black bars below the x-axis indicate a high frequency of ECP treatment during the first 12 weeks (twice per week) followed by a gray bar representing a reduced frequency (twice every second week) in weeks 13–28 Neither frequency nor IFN-γ release of virus specific T cells was hampered by ECP (Figures 6A–C). Besides these, no significant influence of ECP therapy on CD4+CD8+ T cells, γδ T cells, and NKT cells as other well-established protective cell subsets could be observed (Figure 6D). Furthermore, no significant change in NK activity was shown during ECP (Figure 6E) Impact of ECP therapy on anti-viral and anti-leukemic immune responses (A) The representative dot plots with the frequency of CMV-specific CD8+ T cells are shown at different time (T) points before (T1) and after (T2 and T3) ECP treatment in aGvHD patient #5 and cGvHD patient #13 and TE within the CMV specific CD8+ T cells in patients #5 and #13 is indicated (C) The secretion of IFN-γ by virus specific T cells was measured by IFN-γ ELISpot assay The bar chart shows the overview of the INF-γ secretion by CD8+ T cells in seven patients under ECP treatment There is no significant difference among T1 the frequency of CMV-specific CD8+ T cells was maintained Most cells were TE cells followed by TEM cells The function of these cells in terms of IFN-γ release kept stable (D) The dynamic changes of CD4+CD8+ T cells γδ T cells and NKT cells in aGvHD (upper panel) and cGvHD patients (lower panel) under the ECP treatment Cell frequencies were not significantly different between before and under ECP treatment groups (E) A 4-h 51Cr release assay was performed to test the NK activity which was calculated by the following formula: % specific lysis = [c.p.m The box chart shows the NK activity against K562 cells at two different time points in aGvHD group and cGvHD group Each box represents three independent patients The NK cell function was stable over the time of ECP treatment The dashed lines represent the corresponding median value of frequencies observed in 25 healthy donors a p < 0.05 was considered to be statistically significant Cell population dynamics are displayed with steroid dosing and clinical parameters in representative patients under ECP therapy Activated MDSCs subset was steadily increased under ECP therapy in patient #7 The frequency of DN T cells was apparently elevated under ECP treatment (patient #7: 1.2-fold) Similar changes were detected for vδ2+ T cells (patient #7: 2-fold) a strong up-regulation of CD4+ TE cells with a dramatical loss of homing marker CD62L was observed under therapy The frequency of MDSCs and Tregs in patient #16 was stable over time a rebound of Th17 after steroid reduction could be decreased when ECP was continued after allo-HSCT the major goal is to control the activation of alloreactive T cells and to mitigate GvHD through the administration of immunosuppressive drugs we assumed CD19hiCD20hi B cells might play a role in the cGvHD pathophysiology the hypothesis was confirmed by our finding that a reduction of CD19hiCD20hi B cells was observed in patients with clinical improvement under ECP treatment Our data show a significant increase of Bregs in aGvHD patients Immunosuppressive MDSCs, especially the neutrophilic subgroup, can be augmented by ECP and thereby hamper Th1 and Th17 cells through metabolism of L-arginine (12) an increase in monocytic MDSCs was observed in aGvHD patients our data further demonstrate that ECP may promote the development of MDSCs by switching the inactivated (CD33−CD11b+CD14+HLA-DR−/low) to activated (CD33+CD11b+CD14+HLA-DR−/low) subsets In activated MDSCs we observed an up-regulation of the suppressive FAS gene together with an enrichment of the pathway related to negative regulation of immune system process It suggests these activated cells have a more potent immunosuppressive capacity thus contributing to immune tolerance steroids were insufficient to reduce proinflammatory cytokines in all cases since some patients still had high levels of cytokines under the high-dose of immunosuppressive medication ECP has a potent effect on down-modulation of proinflammatory cytokines In line with previous studies (64, 65) our patients showed neither increased susceptibility to infections the frequency of cytotoxic CD8+ T cells the most important mediators of GvL activity and NKT cells remained constant under ECP therapy CMV specific CD8+ T cells were maintained under ECP ECP did neither affect NK activity nor the capacity of virus-specific CD8+ T cells to produce IFN-γ Our results suggest that ECP therapy preserves immunity against infections as well as the GvL effect via maintaining the quality and quantity of effector cells Schematic overview of mechanisms of immunomodulation in aGvHD patients under ECP therapy involving (i) apheresis with ex vivo collection of peripheral mononuclear cells (ii) photoactivation with exposure of leukocyte-enriched plasma to the photosensitizing agent 8-methoxypsoralen and ultraviolet A light which results in crosslinking of the pyrimidine bases in DNA leading to cell death through apoptosis (iii) reinfusion of the ECP-treated cells to the patient (B) Apoptosis of ECP-treated cells play a key role in vivo Engulfing these apoptotic cells by immature dendritic cells results in a tolerogenic phenotype and promotes tolerance through the secretion of immunosuppressive cytokines such as IL-10 and TGF-β as well double negative (DN) T cells and Bregs result in an overall increase in immune tolerance accompanied by a decrease of immune effector cells like IL-17+ T cells and Th1 cells as well as education of TE/EM cells via decreasing CD62L expression ECP promotes the NK cell differentiation from CD56bri to CD56dim NK cells with loss of expression of NKG2D and CD62L ECP constitutes an effective immunomodulatory therapy for both aGvHD and cGvHD without hampering anti-viral and anti-leukemic effects and WK discussed the organization of the manuscript All authors critically reviewed the manuscript PW Honoraria and membership on Advisory Boards for Sanofi-Aventis This Investigator Initiated Research was funded by Therakos The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest Rieck-Wahl for ECP excellent treatment assistance Lindner for excellent technical assistance the peptide facility at the German Cancer Research Center for providing synthetic peptides NIH for providing monomer to construct tetramer and the microarray unit of the DKFZ Genomics and Proteomics Core Facility for providing the Affymetrix GeneChip and related services LW was supported by a scholarship from China Scholarship Council We acknowledge financial support by Deutsche Forschungsgemeinschaft within the funding programme Open Access Publishing by the Baden-Württemberg Ministry of Science Research and the Arts and by Ruprecht-Karls-Universität Heidelberg The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2018.02207/full#supplementary-material multicenter standardization of acute graft-versus-host disease clinical data collection: a report from the Mount Sinai acute GVHD international consortium Current issues in chronic graft-versus-host disease NIH Consensus development project on criteria for clinical trials in chronic graft-versus-host disease: II 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disease The effect of intensified extracorporeal photochemotherapy on long-term survival in patients with severe acute graft-versus-host disease Combination therapy for graft-versus-host disease prophylaxis with etanercept and extracorporeal photopheresis: results of a phase II clinical trial Biol Blood Marrow Transplant (2016) 22:862–8 Deciphering the mystery: extracorporeal photopheresis in graft-versus-host disease Biol Blood Marrow Transplant (2015) 21:1861–2 National institutes of health consensus development project on criteria for clinical trials in chronic graft-versus-host disease: I the 2014 diagnosis and staging working group report Biol Blood Marrow Transplant (2015) 21:389–401e1 Schmitt M and Schmitt A (2018) Modulation of B Cells and Homing Marker on NK Cells Through Extracorporeal Photopheresis in Patients With Steroid-Refractory/Resistant Graft-Vs.-Host Disease Without Hampering Anti-viral/Anti-leukemic Effects Received: 02 May 2018; Accepted: 05 September 2018; Published: 08 October 2018 Copyright © 2018 Wang, Ni, Hückelhoven-Krauss, Sellner, Hoffmann, Neuber, Luft, Hegenbart, Schönland, Kleist, Sill, Chen, Wuchter, Eckstein, Krüger, Hilgendorf, Yerushalmi, Nagler, Müller-Tidow, Ho, Dreger, Schmitt and Schmitt. 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Log in here Hydrogen Buses – Wrightbus Hydroliners launched by German operator WestVerkehr GmbH Zero-emission bus manufacturer Wrightbus has delivered 12 new hydrogen vehicles to German operator WestVerkehr GmbH The latest order completion of Kite Hydroliner single decks means there are now 43 hydrogen-powered buses on the streets of Germany – with around 130 due to be on the roads by the end of 2025 WestVerkehr GmbH is based in the westernmost district of Germany and will operate the Kite Hydroliners between Heinsberg We are delighted that WestVerkehr is bringing our zero-emission hydrogen buses onto the roads of North Rhine-Westphalia This benefits the environment and ensures that people in the region breathe better air What means just as much to us is the excellent partnership and cooperation with WestVerkehr I would like to sincerely thank Managing Director Udo Winkens and his team “I also want to acknowledge our own excellent engineers and designers in Northern Ireland product development and the sales and service teams We can all be very proud of the Kite Hydroliner.” who cut a red ribbon to mark the official commissioning of the buses at a launch event this week WestVerkehr is underlining its commitment to environmentally friendly Together with the partners of the H2HS consortium we are actively driving forward the transformation of local public transport – innovative District Administrator Stephan Pusch said he was particularly pleased about the commissioning of the vehicles: The whole world is talking about the energy transition Hydrogen-powered public transport is an important building block in our efforts to act as a globally sustainable municipality.” State Secretary at the Federal Ministry for Digital and Transport Affairs added: The purchase of hydrogen buses offers real added value: they are quieter more environmentally and climate-friendly and thus contribute to a higher quality of life and environment for people in our cities and villages The use of new technologies reduces noise levels and pollution we are therefore happy to support WestVerkehr GmbH’s initiative to switch to alternative drive systems with 3.4 million euros This is good news for the citizens of the region as well as for climate and environmental protection.” Wrightbus brought its first buses to mainland Europe in 2024 There are now 31 Kite Hydroliners in operation for Regionalverkehr Köln GmbH (RVK) The company has also placed orders for eight additional hydrogen buses by the end of 2025 Deliveries for Saarbahn GmbH (28 Kite Hydroliners) Cottbusverkehr GmbH with Spree-Neiße-Cottbusverkehr GmbH (46 Kite Hydroliners) and Vestische Straßenbahnen GmbH (5 Kite Hydroliners) are also earmarked across 2025 Wrightbus has also opened a European service centre in Brühl our team of experts maintains and services all types of buses here in the heart of Europe “This includes those from other manufacturers and with different drive types.” The service centre includes a spare parts warehouse in Brühl It is the first step on the way to a comprehensive network of service centres on the continent replicating the network that already exists in the UK With its long range of up to 1,000 kilometres the Kite Hydroliner FCEV is ideal for long journeys The single-deck buses can accommodate up to 90 passengers The buses for WestVerkehr meet the high requirements of the VDV (Association of German Transport Companies) and the European General Safety Regulation (GSR2) READ the latest news shaping the hydrogen market at Hydrogen Central Hydrogen Buses – Wrightbus Hydroliners launched by German operator WestVerkehr GmbH, source Ten eCitaro G fuel cell vehicles with new hydrogen drive mode for the public transport network of the city of Stuttgart Premiere for Stuttgart: Daimler Buses has delivered the first eCitaro G fuel cell vehicles with new.. New to the fleet: SSB introduces fuel cell articulated buses – Mercedes-Benz eCitaro G fuel cell H2 mode on the road in Stuttgart in tens Mercedes-Benz eCitaro G fuel cell H2 mode on the road in Stuttgart in tens.. no gasoline – BYD new hydrogen-powered bus is revolutionizing transportation in the US BYD Hybrid Corporation to roll out a new hydrogen fuel cell.. COPYRIGHT POLICY DISCLAIMER TERMS & CONDITIONS PRIVACY POLICY We love meeting interesting people and making new friends Metrics details The Arabidopsis thaliana Receptor-Like Protein RLP30 contributes to immunity against the fungal pathogen Sclerotinia sclerotiorum Here we identify the RLP30-ligand as a small cysteine-rich protein (SCP) that occurs in many fungi and oomycetes and is also recognized by the Nicotiana benthamiana RLP RE02 RLP30 and RE02 share little sequence similarity and respond to different parts of the native/folded protein some Brassicaceae other than Arabidopsis also respond to a linear SCP peptide instead of the folded protein suggesting that SCP is an eminent immune target that led to the convergent evolution of distinct immune receptors in plants RLP30 shows a second ligand specificity for a SCP-nonhomologous protein secreted by bacterial Pseudomonads tabacum results in quantitatively lower susceptibility to bacterial thus demonstrating that detection of immunogenic patterns by Arabidopsis RLP30 is involved in defense against pathogens from three microbial kingdoms Apart from sensing SCPs from different fungi and oomycetes RLP30 can recognize an SCP-unrelated pattern from Pseudomonas bacteria and confers increased resistance to Nicotiana tabacum plants We further reveal distinct immune-sensing mechanisms for SCPs in Brassicaceae and Solanaceae species Our findings unveil an unanticipated complex relationship between PRRs and PAMPs where a single PRR recognizes PAMPs from microorganisms of three kingdoms and where a single PAMP is detected by phylogenetically distinct PRRs from different plant species a Ethylene accumulation in Arabidopsis Col-0 wild-type plants or an rlp30-2 line complemented with a p35S::RLP30-YFP construct 4 h after treatment with water (mock) cinerea infected area as determined by lesion diameter on day 2 after 24 h-treatment of Col-0 wild-type plants rlp30-2 mutants or the rlp30-2/RLP30-YFP complementation line with water (mock) c Bacterial growth in plants pre-treated with water (mock) or 1 μM SCPSs 24 h before infiltration of Pst DC3000 CFU) were quantified in extracts of leaves 3 days after inoculation d Ethylene accumulation in plants of the Brassicaceae and Solanaceae family 4 h after treatment with water (mock) benthamiana transiently co-expressing SCPSs-myc and either RLP30-GFP or RLP23-GFP Leaf protein extracts (Input) were used for co-immunoprecipitation with GFP-trap beads (IP:GFP) and immunoblotting with tag-specific antibodies data points are indicated as dots (n = 6 for a d; n = 20 for c) and plotted as box plots (center line 1.5 times the interquartile range; error bar Statistically significant differences from mock treatments in the respective plants are indicated (two-sided Student’s t test Source data are provided as a Source data file All assays were performed at least three times with similar results all affect RLP30/SCPSs complex formation and are responsible for the non-functionality of these mutated receptors a Ethylene accumulation in Arabidopsis wild-type plants (Col-0) or indicated mutants 4 h after treatment with water (mock) cinerea (Bc) and Phytophthora infestans (Pi) and transiently transformed with RLP23-GFP The TRV2:GUS construct was used as a control Ethylene production was measured 4 h after treatment with water (mock) c Ethylene accumulation in Arabidopsis rlp30-2 mutants or lines stably expressing RLP30-YFP or RE02-GFP (line 4 and 5) 4 h after treatment with water (mock) Data points are indicated as dots (n = 6) and plotted as box plots (center line Statistically significant differences from control treatments are indicated (two-sided Student’s t test Each experiment was repeated three times with similar results SCPSs recognition is mediated by RE02 in N SCP sensor systems arose independently from convergent evolution Interestingly, the RE02-like subfamily does not include any malvid protein sequences, suggesting that malvids lost RE02-like genes, while in contrast RE02-like genes were copied multiple times in grape vine (Supplementary Fig. 7) immunity against microbial pathogens in malvids might rely more strongly on RLP30-related genes They form a monophyletic clade with other Brassicaceae genes supported by maximum bootstrap support suggesting that they originate from a single gene copy in the lineage leading to Brassicaceae a Schematic representation of SCPSs and derived truncations C1C5, C3C8, C1C4 and C3C5. Cysteine (C) residues are numbered and indicated as red lines, predicted disulfide bridges (DB, http://clavius.bc.edu/~clotelab/DiANNA/) are shown on top napus wild-type plants after 4 h treatment with water (mock) and SCPSs with individual cysteine to serine mutations (C1S to C8S according to cysteine numbering shown in a) (b) or SCPSs truncations depicted in a (c) d Determination of EC50 values of SCPSs and the various SCPSs truncations using ethylene accumulation in A napus plants after treatment with increasing concentrations of recombinant SCPSs versions (produced in Pichia) or synthetic C3C5 (n.d the perception diversity for SCPSs or PG in closely related species within the same plant family could indicate adaptive processes in plant pathogen co-evolution in Brassicaceae a Ethylene accumulation in Col-0 wild-type plants rlp30-2 mutants or an rlp30-2 line complemented with a p35S::RLP30-YFP construct 4 h after treatment with water (mock) 1 μM SCPSs or 1.5 μg/ml SCP-like from Pseudomonas syringae pv b Bacterial growth in Col-0 wild-type plants or the rlp30-2/RLP30-YFP complementation line treated with water (mock) or 1.5 μg/ml SCP-likePsp 24 h before infiltration of Pst DC3000 benthamiana (Nb) plants transiently expressing RLP23-GFP or RLP30-GFP and treated for 4 h with water (mock) Data points are indicated as dots (n = 6 for a c; n = 20 for b) and plotted as box plots (center line Statistically significant differences from Col-0 plants (a) or mock treatments (b c) are indicated (two-sided Student’s t test senses patterns from three microbial kingdoms tabacum wild-type plants (Wt) or two transgenic lines (#49 and #55) stably expressing RLP30-RFP and AtSOBIR1-GFP after 4 h treatment with water (mock) tabacum wild-type plants (Wt) or transgenic RLP30/SOBIR1 lines (#49 and #55) tabacum wild-type plants (Wt) or transgenic RLP30/SOBIR1 lines (left shown are representative leaves) and determination of lesion diameter on day 2 after drop inoculation (right) d Growth of Phytophthora capsici on leaves of N tabacum wild-type plants (Wt) or transgenic RLP30/SOBIR1 lines by determination of lesion size (right) of lesions observed under UV light (left shown are representative leaves) on day 2 after drop inoculation Data points are indicated as dots (n = 6 for a; n = 20 for b Statistically significant differences from wild-type (Wt) plants are indicated (two-sided Student’s t test explaining the different structural requirements of RLP30 and RE02 for SCPSs perception and the ability of RLP30 to recognize a bacterial SCP-like pattern only modestly functions in tobacco without co-expressing AtSOBIR1 we propose that co-expression of heterologous PRR/co-receptor pairs may be deployed to overcome putative incompatibilities of ectopically expressed PRRs this way enhanced broad-spectrum resistance may be engineered as compared to expressing single PRRs alone Solanaceae and Brassica plants were grown in a greenhouse at 23 °C under long-day conditions of 16 h of light and 60–70% humidity and SCPSsC3C5 (SAYTCAAPAKSHLTAESDYWVFYWGNEGVSPGVGST) (GenScript) were prepared as 10 mM stock solutions in 100% dimethyl sulfoxide (DMSO) and diluted in water to the desired concentration before use Partially purified SCFE1 fractions from S. sclerotiorum strain 1946 were used18 The most active fractions from eight rounds of fungal culture and the two-step cation-exchange chromatography purification protocol were pooled and dialyzed overnight in 2 l 25 mM MES at 4 °C in a dialysis membrane (ZelluTrans afterwards vacuum-filtrated through a cellulose acetate membrane (Ciro Manufacturing Corporation pore size: 0.2μm) and loaded with a flow rate of 1 ml/min onto a Source 15S 4.6/100PE column (GE Healthcare) equilibrated with buffer A (50 mM MES bound proteins were eluted with a linear gradient of buffer B (500 mM KCl pH 5.4; 0–50% in 15 column volumes) and 500 μl fractions were collected using automated fractionation Three elicitor-active (B11 to B13, Supplementary Fig. 1b) and two inactive (B9 and B15) fractions of SCFE1 (in 50 mM MES buffer pH 7.0 with varying concentration of KCl depending on the elution time point) were selected for proteomics analysis For each fraction a sample volume of 88 µl was used which corresponded to a total protein content per fraction from 3.9 µg (B9) to 16.0 µg (B13) as determined using the BCA protein assay kit (Thermo Fisher Scientific) Digestion of proteins was carried out by addition of trypsin (proteomics grade Roche) at a 1/50 enzyme/protein ratio (w/w) and incubation at 37 °C overnight Digests were acidified by addition of 0.5% (v/v) formic acid and desalted using self-packed StageTips (three disks per micro-column The peptide eluates were dried to completeness and stored at −80 °C For LC-MS/MS analysis all samples were resuspended in 11 µl 0.1% formic acid in HPLC grade water and 5 µl sample per measurement was injected into the mass spectrometer LC-MS/MS measurements were performed on a nanoLC Ultra1D+ (Eksigent CA) coupled online to a Q-Exactive HF-X mass spectrometer (Thermo Fisher Scientific) Peptides were loaded onto a trap column (ReproSil-pur C18-AQ self-packed) at a flow rate of 5 μl/min in 100% solvent A (0.1% formic acid in HPLC grade water) peptides were transferred to an analytical column (ReproSil Gold C18-AQ self-packed) and separated using a 50 min linear gradient from 4% to 32% of solvent B (0.1% formic acid in acetonitrile and 5% (v/v) DMSO) at 300 nl/min flow rate Both nanoLC solvents contained 5% (v/v) DMSO to boost the nanoESI response of peptides The eluate from the analytical column was sprayed via a stainless-steel emitter (Thermo Fisher Scientific) at a source voltage of 2.2 kV into the mass spectrometer The transfer capillary was heated to 275 °C The Q-Exactive HF-X was operated in data-dependent acquisition (DDA) mode automatically switching between MS1 and MS2 spectrum acquisition MS1 spectra were acquired over a mass-to-charge (m/z) range of m/z 350–1400 at a resolution of 60,000 using a maximum injection time of 45 ms and an AGC target value of 3e6 Up to 18 peptide precursors were isolated (isolation window m/z 1.3 fragmented by high-energy collision-induced dissociation (HCD) using 26% normalized collision energy and analyzed at a resolution of 15,000 with a scan range from m/z 200–2000 or with charge states >6+ were excluded The dynamic exclusion duration of fragmented precursor ions was 25 s Agrobacterium tumefaciens strain GV3101 carrying pBIN-plus-SCPSs was infiltrated into leaves of 4–6-week-old N leaves were vacuum-infiltrated with 0.5 M NaCl 0.4 M Na2HPO4 and 0.4 M NaH2PO4 and the apoplastic wash-fluid containing SCPSs (SCPSs-AWF) was collected by centrifugation at 1500 × g for 20 min and 4 °C pastoris culture medium was achieved by affinity chromatography on 5 ml HisTrapFF column (GE Healthcare; equilibrated in 20 mM Tris-HCl pH 8.0) and elution (buffer gradient 0–500 mM imidazole in equilibration buffer) Recombinant protein expression was verified by concentration determination using the Bradford method and by Western Blot analysis using the anti-His antibody (dilution 1:1000 Pseudomonas strains (Pseudomonas syringae pv Pseudomonas stutzeri DSM10701) were grown in baffled flasks (200 ml medium in 1 l flask) in liquid PDB medium (24 g/l Potato Dextrose Broth Duchefa) for 12 h at 21 °C with 210 rpm shaking The culture medium was centrifuged at 6000 rpm for 20 min and then vacuum-filtrated through a filter paper (MN 615 Macherey-Nagel) and subjected to a two-step cation exchange chromatography protocol using an ÄKTA Explorer FPLC system (GE Healthcare) kept at 4 °C the culture filtrate was loaded onto HiTrap SP FF column(s) (GE Healthcare) equilibrated in buffer A (50 mM MES a 100% elution step with buffer B (500 mM KCl pH 5.4) was performed at a flow rate of 1–2 ml/min and the elution peak was monitored with OD280nm OD254nm and OD214nm and collected manually The collected eluate was dialyzed against 2 l 25 mM MES overnight at 4 °C in a dialysis membrane (ZelluTrans pore size: 0.2 μm) and loaded with a flow rate of 1 ml/min onto a Source 15 S 4.6/100PE column (GE Healthcare) equilibrated with buffer A After washing with buffer A the bound proteins were eluted with a linear gradient of buffer B (0–50% in 10 column volumes) and 500 μl fractions were collected using automated fractionation Proteins were detected by immunoblotting with tag-specific antibodies raised against epitope tags GFP (dilution 1:5000) HA (dilution 1:3000) or Myc (dilution 1:5000 followed by staining with secondary antibodies coupled to horseradish peroxidase and ECL (Cytiva) Lesion sizes were determined after 2 d by determining the average lesion diameter tabacum leaves inoculated with 0.8 cm fresh mycelial plugs of Phytophthora capsici grown on V8 juice agar were clipped and kept in the dark in plastic boxes to obtain high humidity Lesion diameters were measured at 48 hpi using UV light No statistical methods were used to predetermine sample size The investigators were not blinded to allocation during experiments and outcome assessment or as box-and-whisker plots in which the center line indicates the median the bounds of the box indicate the 25th and 75th percentiles and the whiskers indicate 1.5 times the interquartile range graphs present data from a single experiment Data were analyzed with a two-sided Student’s t test using Microsoft Office Excel A summary of statistical analyses is provided in Source data Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article Plant immunity: towards an integrated view of plant-pathogen interactions Thirty years of resistance: zig-zag through the plant immune system Pattern recognition receptors and signaling in plant-microbe interactions Plant cell surface immune receptor complex signaling Receptor like proteins associate with SOBIR1-type of adaptors to form bimolecular receptor kinases One for all: the receptor-associated kinase BAK1 Plant immunity: danger perception and signaling A conserved peptide pattern from a widespread microbial virulence factor triggers pattern-induced immunity in Arabidopsis A Phytophthora sojae glycoside hydrolase 12 protein is a major virulence factor during soybean infection and is recognized as a PAMP A small cysteine-rich protein from two kingdoms of microbes is recognized as a novel pathogen-associated molecular pattern Activity and phylogenetics of the broadly occurring family of microbial Nep1-like proteins Genotyping-by-sequencing-based identification of Arabidopsis pattern recognition receptor RLP32 recognizing proteobacterial translation initiation factor IF1 Distinct immune sensor systems for fungal endopolygalacturonases in closely related Brassicaceae Regulation of cell behaviour by plant receptor kinases: pattern recognition receptors as prototypical models The battle for chitin recognition in plant-microbe interactions Arabidopsis receptor-like protein30 and receptor-like kinase suppressor of BIR1-1/EVERSHED mediate innate immunity to necrotrophic fungi An RLP23-SOBIR1-BAK1 complex mediates NLP-triggered immunity Multiple knockout mutants reveal a high redundancy of phytotoxic compounds contributing to necrotrophic pathogenesis of Botrytis cinerea A receptor-like protein from Nicotiana benthamiana mediates VmE02 PAMP-triggered immunity A metacalibrated time-tree documents the early rise of flowering plant phylogenetic diversity PhyloGenes: an online phylogenetics and functional genomics resource for plant gene function inference A genome-wide functional investigation into the roles of receptor-like proteins in Arabidopsis Snoeck, S., Garcia, A. G. K. & Steinbrenner, A. D. Plant receptor-like proteins (RLPs): structural features enabling versatile immune recognition. Physiol. Mol. Plant Pathol. https://doi.org/10.1016/j.pmpp.2023.102004 (2023) Allosteric receptor activation by the plant peptide hormone phytosulfokine The EDS1-PAD4-ADR1 node mediates Arabidopsis pattern-triggered immunity A domain swap approach reveals a role of the plant wall-associated kinase 1 (WAK1) as a receptor of oligogalacturonides Fungal endopolygalacturonases are recognized as microbe-associated molecular patterns by the Arabidopsis receptor-like protein RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1 The kinase LYK5 is a major chitin receptor in Arabidopsis and forms a chitin-induced complex with related kinase CERK1 Interfamily transfer of a plant pattern-recognition receptor confers broad-spectrum bacterial resistance The pattern-recognition receptor CORE of Solanaceae detects bacterial cold-shock protein Whole-genome sequencing of multiple Arabidopsis thaliana populations The pattern of polymorphism in Arabidopsis thaliana MaxQuant enables high peptide identification rates individualized p.p.b.-range mass accuracies and proteome-wide protein quantification Global quantification of mammalian gene expression control SignalP 6.0 predicts all five types of signal peptides using protein language models A modular plasmid assembly kit for multigene expression gene silencing and silencing rescue in plants Development of series of gateway binary vectors for realizing efficient construction of fusion genes for plant transformation Receptor-like kinase SOBIR1/EVR interacts with receptor-like proteins in plant immunity against fungal infection Avr4 promotes Cf-4 receptor-like protein association with the BAK1/SERK3 receptor-like kinase to initiate receptor endocytosis and plant immunity Comparing Arabidopsis receptor kinase and receptor protein-mediated immune signaling reveals BIK1-dependent differences The BIR2/BIR3-associated phospholipase Dgamma1 negatively regulates plant immunity Basal resistance against bacteria in Nicotiana benthamiana leaves is accompanied by reduced vascular staining and suppressed by multiple Pseudomonas syringae type III secretion system effector proteins EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus Concerted expansion and contraction of immune receptor gene repertoires in plant genomes Phytozome: a comparative platform for green plant genomics UniProt: the Universal Protein Knowledgebase in 2023 MAFFT multiple sequence alignment software version 7: improvements in performance and usability IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era Download references Joosten for SlSOBIR1 and NbSOBIR1 constructs and TRV2::GUS Caterina Brancato for assistance in tobacco transformation and Franziska Hackbarth and Hermine Kienberger for excellent laboratory assistance at the BayBioMS This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) to A.A.G the China Scholarship Council (CSC) to Y.Y and the BMBF-funded de.NBI Cloud within the German Network for Bioinformatics Infrastructure (de.NBI) We acknowledge support from the Open Access Publication Fund of the University of Tübingen These authors contributed equally: Yuankun Yang Bavarian Center for Biomolecular Mass Spectrometry conceived and designed the experiments; Y.Y. All authors discussed the results and commented on the manuscript The authors declare no competing interests Nature Communications thanks the other anonymous reviewer(s) for their contribution to the peer review of this work Download citation DOI: https://doi.org/10.1038/s41467-023-39208-8 Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research As London’s Robin Hood Gardens (pictured) is destroyed despite a high-profile campaign to save it we look at some cherished examples of modernist architecture from the 50s Photograph: Antipin Konstantin/Konstantin Antipin This website is using a security service to protect itself from online attacks The action you just performed triggered the security solution There are several actions that could trigger this block including submitting a certain word or phrase You can email the site owner to let them know you were blocked Please include what you were doing when this page came up and the Cloudflare Ray ID found at the bottom of this page Wrightbus has delivered 12 Kite Hydroliner hydrogen fuel cell-electric single-deck buses to WestVerkehr in Germany and expects to have supplied “around 130” such vehicles in that country by the end of 2025 Their arrival takes to 43 the number delivered to German customers so far The WestVerkehr buses will be used in the towns of Erkelenz Heinsberg and Hückelhoven close to the border with the Netherlands Wrightbus CEO Jean-Marc Gales says he is “delighted” that the customer in Germany “is bringing our zero-emission hydrogen buses onto the roads of North Rhine-Westphalia.” Continues Mr Gales: “This benefits the environment and ensures that people in the region breathe better air I would like to sincerely thank Managing Director Udo Winkens and his team.” Green hydrogen for the WestVerkehr buses will come from a 2MW electrolyser that includes a fuelling station and is part of the H2HS project Electricity is sourced from renewable energy plants 70 tonnes of hydrogen will be produced per year with scope to increase that to 200 tonnes per year Mr Winkens notes that procurement of the Kite Hydroliner buses underlines WestVerkehr’s “commitment to environmentally friendly He continues: “Together with the partners of the H2HS consortium we are actively driving forward the transformation of local public transport – innovative Mr Gales has also acknowledged the manufacturer’s own teams for their work on the project Wrightbus has opened a European service centre in Brühl near Cologne to provide support services our team of experts maintains and services all types of buses here in the heart of Europe,” he continues “This includes those from other manufacturers and with different drive types.” Wrightbus plans to open further member of what it terms a comprehensive network of services centres in mainland Europe “replicating the network that already exists in the UK.” routeone magazine is the indispensable resource for professional UK coach The home of vehicle sales and the latest bus and coach job vacancies routeone connects professional PCV operators with complete and unrivalled news coverage.