Publications by authors named "Ulrik Stervbo"

51 Publications

Superior cellular and humoral immunity toward SARS-CoV-2 reference and alpha and beta VOC strains in COVID-19 convalescent as compared to the prime boost BNT162b2 vaccinated dialysis patients.

Kidney Int 2021 Jul 14. Epub 2021 Jul 14.

Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin Center for Advanced Therapies, Berlin, Germany; Center for Translational Medicine and Immune Diagnostics Laboratory, Medical Department I, Marien Hospital Herne, University Hospital of the Ruhr-University Bochum, Herne, Germany. Electronic address:

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http://dx.doi.org/10.1016/j.kint.2021.07.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8277953PMC
July 2021

Detection of pre-existing SARS-CoV-2-reactive T cells in unexposed renal transplant patients.

J Nephrol 2021 Jul 6. Epub 2021 Jul 6.

Center for Translational Medicine and Immune Diagnostics Laboratory, Medical Department I, Marien Hospital Herne, University Hospital of the Ruhr-University Bochum, Hölkeskampring 40, 44625, Herne, Germany.

Background: Recent data demonstrate potentially protective pre-existing T cells reactive against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in samples of healthy blood donors, collected before the SARS-CoV-2 pandemic. Whether pre-existing immunity is also detectable in immunosuppressed patients is currently not known.

Methods: Fifty-seven patients were included in this case-control study. We compared the frequency of SARS-CoV-2-reactive T cells in the samples of 20 renal transplant (RTx) patients to 20 age/gender matched non-immunosuppressed/immune competent healthy individuals collected before the onset of the SARS-CoV-2 pandemic. Seventeen coronavirus disease 2019 (COVID-19) patients were used as positive controls. T cell reactivity against Spike-, Nucleocapsid-, and Membrane- SARS-CoV-2 proteins were analyzed by multi-parameter flow cytometry. Antibodies were analyzed by neutralization assay.

Results: Pre-existing SARS-CoV-2-reactive T cells were detected in the majority of unexposed patients and healthy individuals. In RTx patients, 13/20 showed CD4 T cells reactive against at least one SARS-CoV-2 protein. CD8 T cells reactive against at least one SARS-CoV-2 protein were demonstrated in 12/20 of RTx patients. The frequency and Th1 cytokine expression pattern of pre-formed SARS-CoV-2 reactive T cells did not differ between RTx and non-immunosuppressed healthy individuals.

Conclusions: This study shows that the magnitude and functionality of pre-existing SARS-CoV-2 reactive T cell in transplant patients is non-inferior compared to the immune competent cohort. Although several pro-inflammatory cytokines were produced by the detected T cells, further studies are required to prove their antiviral protection.
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http://dx.doi.org/10.1007/s40620-021-01092-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8259083PMC
July 2021

The impact of acute diarrhea on the coagulation status of patients with vitamin K antagonists.

Sci Rep 2021 Jun 3;11(1):11726. Epub 2021 Jun 3.

Medical Department I, Marien Hospital Herne, University Hospital of the Ruhr-University Bochum, Hölkeskampring 40, 44625, Herne, Germany.

Acute diarrhea is associated with a reduced absorption of both vitamin K antagonists (VKA) and vitamin K itself. To date, the net effect on the coagulation status of subjects with VKA remains elusive. We performed a systematic retrospective single-center analysis using an electronic data extraction approach to identify subjects with plasmatic anticoagulation (either VKA or direct oral anticoagulant (DOAC)) and diarrhea in a German University Hospital over a period of eight years. Acute diarrhea and complete documentation of coagulation status on admission were defined as inclusion criteria, anticoagulation other than VKA/DOAC and obvious inadherence as exclusion criteria. Subjects with VKA/DOAC admitted for hypertension served as control group. Data extraction yielded 356 subjects with gastrointestinal diagnoses and 198 hypertensive subjects, 55 and 83 of whom fulfilled all in- and exclusion criteria. INR values of subjects with VKA were significantly higher in subjects with diarrhea than in hypertensive controls (4.3 ± 3.7 vs. 2.3 ± 0.7, p < 0.001). The distribution of subjects having INR values lower, higher or within the target range differed significantly among groups with a substantially higher prevalence of overanticoagulation in the diarrhea group (46.4% vs. 14.3%, p < 0.001). In a multinomial logistic regression model, acute diarrhea was significantly associated with overanticoagulation (odds ratio 7.2, 95% confidence interval 2.163-23.921; p < 0.001), whereas age, sex, creatinine, and indication of anticoagulation were not (p > 0.05 each). Acute diarrhea is associated with a highly increased risk for overanticoagulation in patients with VKA. Thus, gastroenteritis necessitates a close monitoring of INR in order to identify subjects needing a temporary pause of VKA therapy.
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http://dx.doi.org/10.1038/s41598-021-91316-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8175677PMC
June 2021

The Magnitude and Functionality of SARS-CoV-2 Reactive Cellular and Humoral Immunity in Transplant Population Is Similar to the General Population Despite Immunosuppression.

Transplantation 2021 Mar 18. Epub 2021 Mar 18.

1Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin-Brandenburg Center for Regenerative Therapies, and Institute of Medical Immunology, Germany 2Center for Translational Medicine and Immune Diagnostics Laboratory, Medical Department I, Marien Hospital Herne, University Hospital of the Ruhr-University Bochum, Germany 3Department of Infectious Diseases, West German Centre of Infectious Diseases, University Hospital Essen, University Duisburg-Essen, Germany 4Department of Anesthesiology, University Hospital Essen, University Duisburg-Essen, Germany 5Department of Immunology, Labor Berlin GmbH, Berlin, Germany 6Ruhr-University Bochum, University Hospital Knappschaftskrankenhaus Bochum, Department of Surgery, Bochum, Germany.

Background: The ability of transplant (Tx)-patients to generate a protective antiviral response under immunosuppression is pivotal in COVID-19 infection. However, analysis of immunity against SARS-COV-2 is currently lacking.

Methods: Here, we analyzed T cell immunity directed against SARS-CoV-2 spike-, membrane-, and nucleocapsid-protein by flow cytometry and spike-specific neutralizing antibodies in ten Tx in comparison to 26 nonimmunosuppressed (non-Tx) COVID-19 patients.

Results: Tx-patients (seven renal, one lung, and two combined pancreas-kidney transplants) were recruited in this study during the acute phase of COVID-19 with a median time after SARS-CoV-2-positivity of 3 and 4 days for non-Tx- and Tx-patients, respectively. Despite immunosuppression, we detected antiviral CD4 T cell-response in 90% of Tx-patients. SARS-CoV-2-reactive CD4 T cells produced multiple pro-inflammatory cytokines, indicating their potential protective capacity. Neutralizing antibody-titers did not differ between groups. SARS-CoV-2-reactive CD8+ T cells targeting membrane- and spike-protein were lower in Tx-patients, albeit without statistical significance. However, frequencies of anti-nucleocapsid-protein-reactive, and anti-SARS-CoV-2 polyfunctional CD8 T cells, were similar between patient cohorts. Tx-patients showed features of a prematurely aged adaptive immune system, but equal frequencies of SARS-CoV-2-reactive memory T cells.

Conclusions: In conclusion, a polyfunctional T cell immunity directed against SARS-CoV-2-proteins as well as neutralizing antibodies can be generated in Tx-patients despite immunosuppression. In comparison to nonimmunosuppressed-patients, no differences in humoral and cellular antiviral-immunity were found. Our data presenting the ability to generate SARS-CoV-2-specific immunity in immunosuppressed patients has implications for the handling of SARS-CoV-2-infected Tx patients and raises hopes for effective vaccination in this cohort.Supplemental Visual Abstract; http://links.lww.com/TP/C186.
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http://dx.doi.org/10.1097/TP.0000000000003755DOI Listing
March 2021

Rapid T cell receptor interaction grouping with ting.

Bioinformatics 2021 May 13. Epub 2021 May 13.

Genome Informatics, Institute of Human Genetics, University of Duisburg-Essen, 45147 Essen, Germany.

Motivation: Clustering T cell receptor repertoire (TCRR) sequences according to antigen specificity is challenging. The previously published tool GLIPH needs several days to weeks for clustering large repertoires, making its use impractical in larger studies. In addition, the methodology used in GLIPH suffers from shortcomings, including non-determinism, potential loss of significant antigen-specific sequences or inclusion of too many unspecific sequences.

Results: We present an algorithm for clustering TCRR sequences that scales efficiently to large repertoires. We clustered 36 real datasets with up to 62 000 unique CDR3β sequences using both an implementation of our method called ting, GLIPH and its successsor GLIPH2. While GLIPH required multiple weeks, ting only needed about one minute for the same task. GLIPH2 is comparably fast, but uses a different grouping paradigm. In addition, we found that in naïve repertoires, where no or very few antigen-specific CDR3 sequences or clusters should exist, our method indeed selects much fewer motifs and produces smaller clusters.

Availability: Our method has been implemented in Python as a tool called ting. It is available from GitHub (https://github.com/FelixMoelder/ting) or PyPI under the MIT license.
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http://dx.doi.org/10.1093/bioinformatics/btab361DOI Listing
May 2021

Robust hepatitis B vaccine-reactive T cell responses in failed humoral immunity.

Mol Ther Methods Clin Dev 2021 Jun 23;21:288-298. Epub 2021 Mar 23.

University Hospital Marien Hospital Herne, Ruhr-University of Bochum, Bochum, Germany.

While virus-specific antibodies are broadly recognized as correlates of protection, virus-specific T cells are important for direct clearance of infected cells. Failure to generate hepatitis B virus (HBV)-specific antibodies is well-known in patients with end-stage renal disease. However, whether and to what extent HBV-specific cellular immunity is altered in this population and how it influences humoral immunity is not clear. To address it, we analyzed HBV-reactive T cells and antibodies in hemodialysis patients post vaccination. 29 hemodialysis patients and 10 healthy controls were enrolled in a cross-sectional study. Using multiparameter flow cytometry, HBV-reactive T cells were analyzed and functionally dissected based on granzyme B, interferon-γ (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2), and IL-4 expression. Importantly, HBV-reactive CD4 T cells were detected not only in all patients with sufficient titers but also in 70% of non-responders. Furthermore, a correlation between the magnitude of HBV-reactive CD4 T cells and post-vaccination titers was observed. In summary, our data showed that HBV-reactive polyfunctional T cells were present in the majority of hemodialysis patients even if humoral immunity failed. Further studies are required to confirm their antiviral capacity. The ability to induce vaccine-reactive T cells paves new ways for improved vaccination and therapy protocols.
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http://dx.doi.org/10.1016/j.omtm.2021.03.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8050104PMC
June 2021

Generation of HBsAg-reactive T- and B-cells following HBV vaccination in serological non-responders under hemodialysis treatment.

Eur J Immunol 2021 May 9;51(5):1278-1281. Epub 2021 Feb 9.

Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin-Brandenburg Center for Regenerative Therapies, Berlin, Germany.

HBV vaccination is recommend for hemodialysis patients, but only 50-60% of the patients show seroconversion. HBV vaccine-induced generation of HBV reactive T and B cells could be detected regardless of their capacity to mount a serological response, indicating that patients without seroconversion are potentially protected by their HBV-reactive T cell pool.
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http://dx.doi.org/10.1002/eji.202048756DOI Listing
May 2021

The intratumoral CXCR3 chemokine system is predictive of chemotherapy response in human bladder cancer.

Sci Transl Med 2021 01;13(576)

Institute of Medical Immunology, Charité - Universitätsmedizin Berlin, D-13353 Berlin, Germany.

Chemotherapy has direct toxic effects on cancer cells; however, long-term cancer control and complete remission are likely to involve CD8 T cell immune responses. To study the role of CD8 T cell infiltration in the success of chemotherapy, we examined patients with muscle invasive bladder cancer (MIBC) who were categorized on the basis of the response to neoadjuvant chemotherapy (NAC). We identified the intratumoral CXCR3 chemokine system (ligands and receptor splice variants) as a critical component for tumor eradication upon NAC in MIBC. Through characterization of CD8 T cells, we found that stem-like T cell subpopulations with abundant CXCR3alt, a variant form of the CXCL11 receptor, responded to CXCL11 in culture as demonstrated by migration and enhanced effector function. In tumor biopsies of patients with MIBC accessed before treatment, CXCL11 abundance correlated with high numbers of tumor-infiltrating T cells and response to NAC. The presence of CXCR3alt and CXCL11 was associated with improved overall survival in MIBC. Evaluation of both CXCR3alt and CXCL11 enabled discrimination between responder and nonresponder patients with MIBC before treatment. We validated the prognostic role of the CXCR3-CXCL11 chemokine system in an independent cohort of chemotherapy-treated and chemotherapy-naïve patients with MIBC from data in TCGA. In summary, our data revealed stimulatory activity of the CXCR3alt-CXCL11 chemokine system on CD8 T cells that is predictive of chemotherapy responsiveness in MIBC. This may offer immunotherapeutic options for targeted activation of intratumoral stem-like T cells in solid tumors.
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http://dx.doi.org/10.1126/scitranslmed.abb3735DOI Listing
January 2021

COVID-19-Induced ARDS Is Associated with Decreased Frequency of Activated Memory/Effector T Cells Expressing CD11a.

Mol Ther 2020 12 8;28(12):2691-2702. Epub 2020 Oct 8.

Center for Translational Medicine and Immune Diagnostics Laboratory, Medical Department I, Marien Hospital Herne, University Hospital of the Ruhr University Bochum, Hölkeskampring 40, 44625 Herne, Germany.

Preventing the progression to acute respiratory distress syndrome (ARDS) in COVID-19 is an unsolved challenge. The involvement of T cell immunity in this exacerbation remains unclear. To identify predictive markers of COVID-19 progress and outcome, we analyzed peripheral blood of 10 COVID-19-associated ARDS patients and 35 mild/moderate COVID-19 patients, not requiring intensive care. Using multi-parametric flow cytometry, we compared quantitative, phenotypic, and functional characteristics of circulating bulk immune cells, as well as SARS-CoV-2 S-protein-reactive T cells between the two groups. ARDS patients demonstrated significantly higher S-protein-reactive CD4 and CD8 T cells compared to non-ARDS patients. Of interest, comparison of circulating bulk T cells in ARDS patients to non-ARDS patients demonstrated decreased frequencies of CD4 and CD8 T cell subsets, with activated memory/effector T cells expressing tissue migration molecule CD11a. Importantly, survival from ARDS (4/10) was accompanied by a recovery of the CD11a T cell subsets in peripheral blood. Conclusively, data on S-protein-reactive polyfunctional T cells indicate the ability of ARDS patients to generate antiviral protection. Furthermore, decreased frequencies of activated memory/effector T cells expressing tissue migratory molecule CD11a observed in circulation of ARDS patients might suggest their involvement in ARDS development and propose the CD11a-based immune signature as a possible prognostic marker.
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http://dx.doi.org/10.1016/j.ymthe.2020.10.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7543694PMC
December 2020

Epitope similarity cannot explain the pre-formed T cell immunity towards structural SARS-CoV-2 proteins.

Sci Rep 2020 11 4;10(1):18995. Epub 2020 Nov 4.

Center for Translational Medicine, University Hospital Marien Hospital Herne, Ruhr-University, Bochum, Germany.

The current pandemic is caused by the SARS-CoV-2 virus and large progress in understanding the pathology of the virus has been made since its emergence in late 2019. Several reports indicate short lasting immunity against endemic coronaviruses, which contrasts studies showing that biobanked venous blood contains T cells reactive to SARS-CoV-2 S-protein even before the outbreak in Wuhan. This suggests a preformed T cell memory towards structural proteins in individuals not exposed to SARS-CoV-2. Given the similarity of SARS-CoV-2 to other members of the Coronaviridae family, the endemic coronaviruses appear likely candidates to generate this T cell memory. However, given the apparent poor immunological memory created by the endemic coronaviruses, immunity against other common pathogens might offer an alternative explanation. Here, we utilize a combination of epitope prediction and similarity to common human pathogens to identify potential sources of the SARS-CoV-2 T cell memory. Although beta-coronaviruses are the most likely candidates to explain the pre-existing SARS-CoV-2 reactive T cells in uninfected individuals, the SARS-CoV-2 epitopes with the highest similarity to those from beta-coronaviruses are confined to replication associated proteins-not the host interacting S-protein. Thus, our study suggests that the observed SARS-CoV-2 pre-formed immunity to structural proteins is not driven by near-identical epitopes.
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http://dx.doi.org/10.1038/s41598-020-75972-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642385PMC
November 2020

Regulatory T cells for minimising immune suppression in kidney transplantation: phase I/IIa clinical trial.

BMJ 2020 10 21;371:m3734. Epub 2020 Oct 21.

Berlin Institute of Health Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, Berlin, Germany

Objective: To assess whether reshaping of the immune balance by infusion of autologous natural regulatory T cells (nTregs) in patients after kidney transplantation is safe, feasible, and enables the tapering of lifelong high dose immunosuppression, with its limited efficacy, adverse effects, and high direct and indirect costs, along with addressing several key challenges of nTreg treatment, such as easy and robust manufacturing, danger of over immunosuppression, interaction with standard care drugs, and functional stability in an inflammatory environment in a useful proof-of-concept disease model.

Design: Investigator initiated, monocentre, nTreg dose escalation, phase I/IIa clinical trial (ONEnTreg13).

Setting: Charité-University Hospital, Berlin, Germany, within the ONE study consortium (funded by the European Union).

Participants: Recipients of living donor kidney transplant (ONEnTreg13, n=11) and corresponding reference group trial (ONErgt11-CHA, n=9).

Interventions: CD4+ CD25+ FoxP3+ nTreg products were given seven days after kidney transplantation as one intravenous dose of 0.5, 1.0, or 2.5-3.0×10 cells/kg body weight, with subsequent stepwise tapering of triple immunosuppression to low dose tacrolimus monotherapy until week 48.

Main Outcome Measures: The primary clinical and safety endpoints were assessed by a composite endpoint at week 60 with further three year follow-up. The assessment included incidence of biopsy confirmed acute rejection, assessment of nTreg infusion related adverse effects, and signs of over immunosuppression. Secondary endpoints addressed allograft functions. Accompanying research included a comprehensive exploratory biomarker portfolio.

Results: For all patients, nTreg products with sufficient yield, purity, and functionality could be generated from 40-50 mL of peripheral blood taken two weeks before kidney transplantation. None of the three nTreg dose escalation groups had dose limiting toxicity. The nTreg and reference groups had 100% three year allograft survival and similar clinical and safety profiles. Stable monotherapy immunosuppression was achieved in eight of 11 (73%) patients receiving nTregs, while the reference group remained on standard dual or triple drug immunosuppression (P=0.002). Mechanistically, the activation of conventional T cells was reduced and nTregs shifted in vivo from a polyclonal to an oligoclonal T cell receptor repertoire.

Conclusions: The application of autologous nTregs was safe and feasible even in patients who had a kidney transplant and were immunosuppressed. These results warrant further evaluation of Treg efficacy and serve as the basis for the development of next generation nTreg approaches in transplantation and any immunopathologies.

Trial Registration: NCT02371434 (ONEnTreg13) and EudraCT:2011-004301-24 (ONErgt11).
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http://dx.doi.org/10.1136/bmj.m3734DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7576328PMC
October 2020

Robust T Cell Response Toward Spike, Membrane, and Nucleocapsid SARS-CoV-2 Proteins Is Not Associated with Recovery in Critical COVID-19 Patients.

Cell Rep Med 2020 Sep 29;1(6):100092. Epub 2020 Aug 29.

Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, BIH Center for Regenerative Therapies, Berlin, Berlin, Germany.

T cell immunity toward SARS-CoV-2 spike (S-), membrane (M-), and nucleocapsid (N-) proteins may define COVID-19 severity. Therefore, we compare the SARS-CoV-2-reactive T cell responses in moderate, severe, and critical COVID-19 patients and unexposed donors. Overlapping peptide pools of all three proteins induce SARS-CoV-2-reactive T cell response with dominance of CD4 over CD8 T cells and demonstrate interindividual immunity against the three proteins. M-protein induces the highest frequencies of CD4 T cells, suggesting its relevance for diagnosis and vaccination. The T cell response of critical COVID-19 patients is robust and comparable or even superior to non-critical patients. Virus clearance and COVID-19 survival are not associated with either SARS-CoV-2 T cell kinetics or magnitude of T cell responses, respectively. Thus, our data do not support the hypothesis of insufficient SARS-CoV-2-reactive immunity in critical COVID-19. Conversely, it indicates that activation of differentiated memory effector T cells could cause hyperreactivity and immunopathogenesis in critical patients.
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http://dx.doi.org/10.1016/j.xcrm.2020.100092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7456276PMC
September 2020

Cytocompatibility Evaluation of a Novel Series of PEG-Functionalized Lactide-Caprolactone Copolymer Biomaterials for Cardiovascular Applications.

Front Bioeng Biotechnol 2020 13;8:991. Epub 2020 Aug 13.

Salber Laboratory, Centre for Clinical Research, Department of Experimental Surgery, Ruhr-Universität Bochum, Bochum, Germany.

Although the use of bioresorbable materials in stent production is thought to improve long-term safety compared to their durable counterparts, a recent FDA report on the 2-year follow-up of the first FDA-approved bioresorbable vascular stent showed an increased occurrence of major adverse cardiac events and thrombosis in comparison to the metallic control. In order to overcome the issues of first generation bioresorbable polymers, a series of polyethylene glycol-functionalized poly-L-lactide-co-ε-caprolactone copolymers with varying lactide-to-caprolactone content is developed using a novel one-step PEG-functionalization and copolymerization strategy. This approach represents a new facile way toward surface enhancement for cellular interaction, which is shown by screening these materials regarding their cyto- and hemocompatibility in terms of cytotoxicity, hemolysis, platelet adhesion, leucocyte activation and endothelial cell adhesion. By varying the lactide-to-caprolactone polymer composition, it is possible to gradually affect endothelial and platelet adhesion which allows fine-tuning of the biological response based on polymer chemistry. All polymers developed were non-cytotoxic, had acceptable leucocyte activation levels and presented non-hemolytic (<2% hemolysis rate) behavior except for PLCL-PEG 55:45 which presented hemolysis rate of 2.5% ± 0.5. Water contact angles were reduced in the polymers containing PEG functionalization (PLLA-PEG: 69.8° ± 2.3, PCL-PEG: 61.2° ± 7.5) versus those without (PLLA: 79.5° ± 3.2, PCL: 76.4° ± 10.2) while the materials PCL-PEG550, PLCL-PEG550 90:10 and PLCL-PEG550 70:30 demonstrated best endothelial cell adhesion. PLLA-PEG550 and PLCL-PEG550 70:30 presented as best candidates for cardiovascular implant use from a cytocompatibility perspective across the spectrum of testing completed. Altogether, these polymers are excellent innovative materials suited for an application in stent manufacture due to the ease in translation of this one-step synthesis strategy to device production and their excellent cyto- and hemocompatibility.
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http://dx.doi.org/10.3389/fbioe.2020.00991DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7438451PMC
August 2020

Lessons for the clinical nephrologist: recurrence of nephrotic syndrome induced by SARS-CoV-2.

J Nephrol 2020 Dec 5;33(6):1369-1372. Epub 2020 Sep 5.

Medical Department 1, University Hospital Marien Hospital Herne, Ruhr-University Bochum, Hölkeskampring 40, 44625, Herne, Germany.

SARS-CoV-2 is characterized by a multiorgan tropism including the kidneys. Recent autopsy series indicated that SARS-CoV-2 can infect both tubular and glomerular cells. Whereas tubular cell infiltration may contribute to acute kidney injury, data on a potential clinical correlative to glomerular affection is rare. We describe the first case of nephrotic syndrome in the context of COVID-19 in a renal transplant recipient. A 35 year old male patient received a kidney allograft for primary focal segmental glomerulosclerosis (FSGS). Three months posttransplant a recurrence of podocytopathy was successfully managed by plasma exchange, ivIG, and a conversion from tacrolimus to belatacept (initial proteinuria > 6 g/l decreased to 169 mg/l). Six weeks later he was tested positive for SARS-CoV-2 and developed a second increase of proteinuria (5.6 g/l). Renal allograft biopsy revealed diffuse podocyte effacement and was positive for SARS-CoV-2 in RNA in-situ hybridation indicating a SARS-CoV-2 associated recurrence of podocytopathy. Noteworthy, nephrotic proteinuria resolved spontaneously after recovering from COVID-19. The present case expands the spectrum of renal involvement in COVID-19 from acute tubular injury to podocytopathy in renal transplant recipients. Thus, it may be wise to test for SARS-CoV-2 prior to initiation of immunosuppression in new onset glomerulopathy during the pandemic.
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http://dx.doi.org/10.1007/s40620-020-00855-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7474570PMC
December 2020

Immune monitoring facilitates the clinical decision in multifocal COVID-19 of a pancreas-kidney transplant patient.

Am J Transplant 2020 11 9;20(11):3210-3215. Epub 2020 Sep 9.

Medical Department, University Hospital Marien Hospital Herne, Ruhr-University, Bochum, Germany.

The optimal management in transplant recipients with coronavirus disease 2019 (COVID-19) remains uncertain. The main concern is the ability of immunosuppressed patients to generate sufficient immunity for antiviral protection. Here, we report on immune monitoring facilitating a successful outcome of severe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-associated pneumonia, meningoencephalitis, gastroenteritis, and acute kidney and pancreas graft failure in a pancreas-kidney transplant recipient. Despite the very low numbers of circulating B, NK, and T cells identified in follow-up, a strong SARS-CoV-2 reactive T cell response was observed. Importantly, we detected T cells reactive to Spike, Membrane, and Nucleocapsid proteins of SARS-CoV-2 with majority of T cells showing polyfunctional proinflammatory Th1 phenotype at all analyzed time points. Antibodies against Spike protein were also detected with increasing titers in follow-up. Neutralization tests confirmed their antiviral protection. A correlation between cellular and humoral immunity was observed underscoring the specificity of demonstrated data. We conclude that analyzing the kinetics of nonspecific and SARS-CoV-2-reactive cellular and humoral immunity can facilitate the clinical decision on immunosuppression adjustment and allow successful outcome as demonstrated in the current clinical case. Although the antiviral protection of the detected SARS-CoV-2-reactive T cells requires further evaluation, our data prove an ability mounting a strong SARS-CoV-2-reactive T cell response with functional capacity in immunosuppressed patients.
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http://dx.doi.org/10.1111/ajt.16252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7436473PMC
November 2020

Allograft infiltration and meningoencephalitis by SARS-CoV-2 in a pancreas-kidney transplant recipient.

Am J Transplant 2020 11 30;20(11):3216-3220. Epub 2020 Aug 30.

Medical Department I, University Hospital Marien Hospital Herne, Ruhr-University Bochum, Bochum, Germany.

Severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) preferentially affects epithelia of the upper and lower respiratory tract. Thus, impairment of kidney function has been primarily attributed until now to secondary effects such as cytokine release or fluid balance disturbances. We provide evidence that SARS-CoV-2 can directly infiltrate a kidney allograft. A 69-year-old male, who underwent pancreas-kidney transplantation 13 years previously, presented to our hospital with coronavirus disease 2019 (COVID-19) pneumonia and impaired pancreas and kidney allograft function. Kidney biopsy was performed showing tubular damage and an interstitial mononuclear cell infiltrate. Reverse transcriptase polymerase chain reaction from the biopsy specimen was positive for SARS-CoV-2. In-situ hybridization revealed SARS-CoV-2 RNA in tubular cells and the interstitium. Subsequently, he had 2 convulsive seizures. Magnetic resonance tomography suggested meningoencephalitis, which was confirmed by SARS-CoV-2 RNA transcripts in the cerebrospinal fluid. The patient had COVID-19 pneumonia, meningoencephalitis, and nephritis. SARS-CoV-2 binds to its target cells through angiotensin-converting enzyme 2, which is expressed in a broad variety of tissues including the lung, brain, and kidney. SARS-CoV-2 thereby shares features with other human coronaviruses including SARS-CoV that were identified as pathogens beyond the respiratory tract as well. The present case should provide awareness that extrapulmonary symptoms in COVID-19 may be attributable to viral infiltration of diverse organs.
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http://dx.doi.org/10.1111/ajt.16223DOI Listing
November 2020

Nuclear antigen-reactive CD4 T cells expand in active systemic lupus erythematosus, produce effector cytokines, and invade the kidneys.

Kidney Int 2021 01 24;99(1):238-246. Epub 2020 Jun 24.

Department of Nephrology and Medical Intensive Care, Charité - Universitätsmedizin Berlin, Berlin, Germany. Electronic address:

Systemic lupus erythematosus is a systemic and chronic autoimmune disease characterized by loss of tolerance towards nuclear antigens with autoreactive CD4 T cells implicated in disease pathogenesis. However, very little is known about their receptor specificity since the detection of human autoantigen specific CD4 T cells has been extremely challenging. Here we present an analysis of CD4 T cells reactive to nuclear antigens using two complementary methods: T cell libraries and antigen-reactive T cell enrichment. The frequencies of nuclear antigen specific CD4 T cells correlated with disease severity. These autoreactive T cells produce effector cytokines such as interferon-γ, interleukin-17, and interleukin-10. Compared to blood, these cells were enriched in the urine of patients with active lupus nephritis, suggesting an infiltration of the inflamed kidneys. Thus, these previously unrecognized characteristics support a role for nuclear antigen-specific CD4 T cells in systemic lupus erythematosus.
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http://dx.doi.org/10.1016/j.kint.2020.05.051DOI Listing
January 2021

Discrete populations of isotype-switched memory B lymphocytes are maintained in murine spleen and bone marrow.

Nat Commun 2020 05 22;11(1):2570. Epub 2020 May 22.

Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, 10117, Berlin, Germany.

At present, it is not clear how memory B lymphocytes are maintained over time, and whether only as circulating cells or also residing in particular tissues. Here we describe distinct populations of isotype-switched memory B lymphocytes (Bsm) of murine spleen and bone marrow, identified according to individual transcriptional signature and B cell receptor repertoire. A population of marginal zone-like cells is located exclusively in the spleen, while a population of quiescent Bsm is found only in the bone marrow. Three further resident populations, present in spleen and bone marrow, represent transitional and follicular B cells and B1 cells, respectively. A population representing 10-20% of spleen and bone marrow memory B cells is the only one qualifying as circulating. In the bone marrow, all cells individually dock onto VCAM1 stromal cells and, reminiscent of resident memory T and plasma cells, are void of activation, proliferation and mobility.
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http://dx.doi.org/10.1038/s41467-020-16464-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7244721PMC
May 2020

Differential Diagnosis of Interstitial Allograft Rejection and BKV Nephropathy by T-cell Receptor Sequencing.

Transplantation 2020 04;104(4):e107-e108

Medical Department I, Universitätsklinikum der Ruhr-Universität Bochum, Ruhr-Universität Bochum, Herne, Germany.

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http://dx.doi.org/10.1097/TP.0000000000003054DOI Listing
April 2020

Propionate supplementation promotes the expansion of peripheral regulatory T-Cells in patients with end-stage renal disease.

J Nephrol 2020 Aug 6;33(4):817-827. Epub 2020 Mar 6.

Medical Department I , Centre for Translational Medicine, Marienhospital Herne, Universitätsklinikum Der Ruhr-Universität Bochum, Ruhr-Universität Bochum, Hölkeskampring 40, 44623, Herne, Germany.

Patients with end-stage renal disease (ESRD) suffer from a progressively increasing low-grade systemic inflammation, which is associated with higher morbidity and mortality. Regulatory T cells (Tregs) play an important role in regulation of the inflammatory process. Previously, it has been demonstrated that short-chain fatty acids reduce inflammation in the central nervous system in a murine model of multiple sclerosis through an increase in tissue infiltrating Tregs. Here, we evaluated the effect of the short-chain fatty acid propionate on the chronic inflammatory state and T-cell composition in ESRD patients. Analyzing ESRD patients and healthy blood donors before, during, and 60 days after the propionate supplementation by multiparametric flow cytometry we observed a gradual and significant expansion in the frequencies of CD25CD127 Tregs in both groups. Phenotypic characterization suggests that polarization of naïve T cells towards Tregs is responsible for the observed expansion. In line with this, we observed a significant reduction of inflammatory marker CRP under propionate supplementation. Of interest, the observed anti-inflammatory surroundings did not affect the protective pathogen-specific immunity as demonstrated by the stable frequencies of effector/memory T cells specific for tetanus/diphtheria recall antigens. Collectively, our data suggest that dietary supplements with propionate have a beneficial effect on the elevated systemic inflammation of ESRD patients. The effect can be achieved through an expansion of circulating Tregs without affecting the protective pathogen-reactive immunity.
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http://dx.doi.org/10.1007/s40620-019-00694-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7381474PMC
August 2020

Stability of 12 T-helper cell-associated cytokines in human serum under different pre-analytical conditions.

Cytokine 2020 05 25;129:155044. Epub 2020 Feb 25.

Center for Translational Medicine, Medical Department I, Marien Hospital Herne, University Hospital of the Ruhr-University Bochum, Hölkeskampring 40, 44625 Herne, Germany; Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin-Brandenburg Center for Regenerative Therapies, Augustenburger Platz 1, 13353 Berlin, Germany. Electronic address:

Cytokines are soluble and readily analyzed signaling molecules which reveal vital cues about the state of the immune system. As such, they serve in diagnosis and monitoring of immune-related disorders, where strictly controlled handling of the samples including storage and freeze/thawing procedures are required. In basic research and clinical trials, human serum samples can be left for long-term storage before processing. Storage space is commonly limited in scientific laboratories, which require storage of fewer but larger aliquots of patient serum samples. There are also practical limitations to the number of analytes to be processed at the same time. Further, new findings and technological progress might prompt analysis of hitherto unconsidered or undetectable molecules. Repeated freeze/thawing of serum samples is therefore a likely scenario, raising the question of the stability of the measured analytes under such conditions. To address this question, we subjected serum samples with spiked-in T-helper cell associated cytokines to several cycles of freeze/thawing under different conditions, including storage at -20 °C or -80 °C and thawing at 4 °C, 22 °C, and 37 °C, respectively. The concentration of TNF-α, IL-4, IL-17F, and IL-22 decreased after storage at room temperature for 4 h before freezing. Generally, storage at -20 °C resulted in reduced cytokine concentrations. This contrasts storage at -80 °C, which gave stable analyte concentrations; unaffected by repeated freeze/thaw cycles. The study presented here highlights the need for sentinel samples with known cytokine concentrations as internal control for the freeze/thaw process.
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http://dx.doi.org/10.1016/j.cyto.2020.155044DOI Listing
May 2020

The TreaT-Assay: A Novel Urine-Derived Donor Kidney Cell-Based Assay for Prediction of Kidney Transplantation Outcome.

Sci Rep 2019 12 13;9(1):19037. Epub 2019 Dec 13.

Berlin Institute of Health Center for Regenerative Therapies (BCRT), Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.

Donor-reactive immunity plays a major role in rejection after kidney transplantation, but analysis of donor-reactive T-cells is not applied routinely. However, it has been shown that this could help to identify patients at risk of acute rejection. A major obstacle is the limited quantity or quality of the required allogenic stimulator cells, including a limited availability of donor-splenocytes or an insufficient HLA-matching with HLA-bank cells. To overcome these limitations, we developed a novel assay, termed the TreaT (Transplant reactive T-cells)-assay. We cultivated renal tubular epithelial cells from the urine of kidney transplant patients and used them as stimulators for donor-reactive T-cells, which we analyzed by flow cytometry. We could demonstrate that using the TreaT-assay the quantification and characterization of alloreactive T-cells is superior to other stimulators. In a pilot study, the number of pre-transplant alloreactive T-cells negatively correlated with the post-transplant eGFR. Frequencies of pre-transplant CD161 alloreactive CD4 T-cells and granzyme B producing alloreactive CD8 T-cells were substantially higher in patients with early acute rejection compared to patients without complications. In conclusion, we established a novel assay for the assessment of donor-reactive memory T-cells based on kidney cells with the potential to predict early acute rejection and post-transplant eGFR.
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http://dx.doi.org/10.1038/s41598-019-55442-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6911059PMC
December 2019

VDJdb in 2019: database extension, new analysis infrastructure and a T-cell receptor motif compendium.

Nucleic Acids Res 2020 01;48(D1):D1057-D1062

Pirogov Russian Medical State University, Moscow, Russia.

Here, we report an update of the VDJdb database with a substantial increase in the number of T-cell receptor (TCR) sequences and their cognate antigens. The update further provides a new database infrastructure featuring two additional analysis modes that facilitate database querying and real-world data analysis. The increased yield of TCR specificity identification methods and the overall increase in the number of studies in the field has allowed us to expand the database more than 5-fold. Furthermore, several new analysis methods are included. For example, batch annotation of TCR repertoire sequencing samples allows for annotating large datasets on-line. Using recently developed bioinformatic methods for TCR motif mining, we have built a reduced set of high-quality TCR motifs that can be used for both training TCR specificity predictors and matching against TCRs of interest. These additions enhance the versatility of the VDJdb in the task of exploring T-cell antigen specificities. The database is available at https://vdjdb.cdr3.net.
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http://dx.doi.org/10.1093/nar/gkz874DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6943061PMC
January 2020

Killer-like receptors and GPR56 progressive expression defines cytokine production of human CD4 memory T cells.

Nat Commun 2019 05 22;10(1):2263. Epub 2019 May 22.

Institute of Medical Immunology, Charité - Universitätsmedizin Berlin, Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, 13353, Berlin, Germany.

All memory T cells mount an accelerated response on antigen reencounter, but significant functional heterogeneity is present within the respective memory T-cell subsets as defined by CCR7 and CD45RA expression, thereby warranting further stratification. Here we show that several surface markers, including KLRB1, KLRG1, GPR56, and KLRF1, help define low, high, or exhausted cytokine producers within human peripheral and intrahepatic CD4 memory T-cell populations. Highest simultaneous production of TNF and IFN-γ is observed in KLRB1KLRG1GPR56 CD4 T cells. By contrast, KLRF1 expression is associated with T-cell exhaustion and reduced TNF/IFN-γ production. Lastly, TCRβ repertoire analysis and in vitro differentiation support a regulated, progressive expression for these markers during CD4 memory T-cell differentiation. Our results thus help refine the classification of human memory T cells to provide insights on inflammatory disease progression and immunotherapy development.
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http://dx.doi.org/10.1038/s41467-019-10018-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6531457PMC
May 2019

Repeated Changes to the Gravitational Field Negatively Affect the Serum Concentration of Select Growth Factors and Cytokines.

Front Physiol 2019 17;10:402. Epub 2019 Apr 17.

Center for Translational Medicine, Medical Department I, Marien Hospital Herne, University Hospitals of the Ruhr-University of Bochum, Herne, Germany.

Space flights, some physical activities, and extreme sports can greatly alter the gravitational forces experienced by the body. Being a deviation from the constant pull of Earth, these alterations can be considered gravitational stress and have the potential to affect physiological processes. Physical cues play a vital role in the homeostasis and function of the immune system. The effect of recurrent alterations of the gravitational pull on the levels of soluble mediator such as cytokines is unknown. Parabolic flights provide a controlled environment and make these a suitable model to study the effects of gravitational stress. Utilizing this model, we evaluated the effects of short-term gravitational stress on serum concentration of cytokines and other soluble mediators. Blood was taken from 12 healthy volunteers immediately before the first parabola and immediately after the last. Samples taken on the ground at corresponding time points the day before were used to control for circadian effects. A wide range of soluble mediators was analyzed using a multiplex bead assay. We found that the rate-change of eight molecules was significantly affected by the parabolic flight. Among other functions, these molecules, EGF, PDGF-AA, PDGF-BB, HGF, IP-10, Eotaxin (CCL11), TARC, and Angiopoietin-2, can be associated with bone remodeling and immune activation. It is therefore possible that gravitational stress can have clinically relevant impact on the control of a wide range of physiological processes.
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http://dx.doi.org/10.3389/fphys.2019.00402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6478750PMC
April 2019

The Identity Card of T Cells-Clinical Utility of T-cell Receptor Repertoire Analysis in Transplantation.

Transplantation 2019 08;103(8):1544-1555

Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, Berlin, Germany.

There is a clear medical need to change the current strategy of "one-size-fits-all" immunosuppression for controlling transplant rejection to precision medicine and targeted immune intervention. As T cells play a key role in both undesired graft rejection and protection, a better understanding of the fate and function of both alloreactive graft-deteriorating T cells and those protecting to infections is required. The T-cell receptor (TCR) is the individual identity card of each T cell clone and can help to follow single specificities. In this context, tracking of lymphocytes with certain specificity in blood and tissue in clinical follow up is of especial importance. After overcoming technical limitations of the past, novel molecular technologies opened new avenues of diagnostics. Using advantages of next generation sequencing, a method was established for T-cell tracing by detection of variable TCR region as identifiers of individual lymphocyte clones. The current review describes principles of laboratory and computational methods of TCR repertoire analysis, and gives an overview on applications for the basic understanding of transplant biology and immune monitoring. The review also delineates methodological pitfalls and challenges. With the outlook on prediction of antigens in immune-mediated processes including those of unknown causative pathogens, monitoring the fate and function of individual T cell clones, and the adoptive transfer of protective effector or regulatory T cells, this review highlights the current and future capability of TCR repertoire analysis.
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http://dx.doi.org/10.1097/TP.0000000000002776DOI Listing
August 2019

BKV Clearance Time Correlates With Exhaustion State and T-Cell Receptor Repertoire Shape of BKV-Specific T-Cells in Renal Transplant Patients.

Front Immunol 2019 10;10:767. Epub 2019 Apr 10.

Center for Translational Medicine, Medical Clinic I, Marien Hospital Herne, University Hospital of the Ruhr-University Bochum, Herne, Germany.

Reactivation of the BK polyomavirus is known to lead to severe complications in kidney transplant patients. The current treatment strategy relies on decreasing the immunosuppression to allow the immune system to clear the virus. Recently, we demonstrated a clear association between the resolution of BKV reactivation and reconstitution of BKV-specific CD4 T-cells. However, which factors determine the duration of viral infection clearance remains so far unclear. Here we apply a combination of in-depth multi-parametric flow cytometry and NGS-based CDR3 beta chain receptor repertoire analysis of BKV-specific T-cells to a cohort of 7 kidney transplant patients during the clinical course of BKV reactivation. This way we followed TCR repertoires at single clone levels and functional activity of BKV-specific T-cells during the resolution of BKV infection. The duration of BKV clearance did not depend on the number of peripheral blood BKV-specific T-cells nor on a few immunodominant BKV-specific T-cell clones. Rather, the T-cell receptor repertoire diversity and exhaustion status of BKV-specific T-cells affected the duration of viral clearance: high clonotype diversity and lack of PD1 and TIM3 exhaustion markers on BKV-specific T-cells was associated with short clearance time. Our data thus demonstrate how the diversity and the exhaustion state of the T-cells can determine the clinical course of BKV infection.
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http://dx.doi.org/10.3389/fimmu.2019.00767DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468491PMC
September 2020
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