Publications by authors named "Stephan Schlickeiser"

36 Publications

Disease Severity, Fever, Age, and Sex Correlate With SARS-CoV-2 Neutralizing Antibody Responses.

Front Immunol 2020 29;11:628971. Epub 2021 Jan 29.

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

Clinical trials on the use of COVID-19 convalescent plasma remain inconclusive. While data on safety is increasingly available, evidence for efficacy is still sparse. Subgroup analyses hint to a dose-response relationship between convalescent plasma neutralizing antibody levels and mortality. In particular, patients with primary and secondary antibody deficiency might benefit from this approach. However, testing of neutralizing antibodies is limited to specialized biosafety level 3 laboratories and is a time- and labor-intense procedure. In this single center study of 206 COVID-19 convalescent patients, clinical data, results of commercially available ELISA testing of SARS-CoV-2 spike-IgG and -IgA, and levels of neutralizing antibodies, determined by plaque reduction neutralization testing (PRNT), were analyzed. At a medium time point of 58 days after symptom onset, only 12.6% of potential plasma donors showed high levels of neutralizing antibodies (PRNT50 ≥ 1:320). Multivariable proportional odds logistic regression analysis revealed need for hospitalization due to COVID-19 (odds ratio 6.87; -value 0.0004) and fever (odds ratio 3.00; -value 0.0001) as leading factors affecting levels of SARS-CoV-2 neutralizing antibody titers in convalescent plasma donors. Using penalized estimation, a predictive proportional odds logistic regression model including the most important variables hospitalization, fever, age, sex, and anosmia or dysgeusia was developed. The predictive discrimination for PRNT50 ≥ 1:320 was reasonably good with AUC: 0.86 (with 95% CI: 0.79-0.92). Combining clinical and ELISA-based pre-screening, assessment of neutralizing antibodies could be spared in 75% of potential donors with a maximal loss of 10% of true positives (PRNT50 ≥ 1:320).
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http://dx.doi.org/10.3389/fimmu.2020.628971DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878374PMC
February 2021

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

Sci Transl Med 2021 Jan;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

Feasibility, long-term safety, and immune monitoring of regulatory T cell therapy in living donor kidney transplant recipients.

Am J Transplant 2021 04 2;21(4):1603-1611. Epub 2021 Feb 2.

Peter Gorer Department of Immunobiology, School of Immunology and Microbial Science, Kings College London, London, UK.

Short-term outcomes in kidney transplantation are marred by progressive transplant failure and mortality secondary to immunosuppression toxicity. Immune modulation with autologous polyclonal regulatory T cell (Treg) therapy may facilitate immunosuppression reduction promoting better long-term clinical outcomes. In a Phase I clinical trial, 12 kidney transplant recipients received 1-10 × 10 Treg per kg at Day +5 posttransplantation in lieu of induction immunosuppression (Treg Therapy cohort). Nineteen patients received standard immunosuppression (Reference cohort). Primary outcomes were rejection-free and patient survival. Patient and transplant survival was 100%; acute rejection-free survival was 100% in the Treg Therapy versus 78.9% in the reference cohort at 48 months posttransplant. Treg therapy revealed no excess safety concerns. Four patients in the Treg Therapy cohort had mycophenolate mofetil withdrawn successfully and remain on tacrolimus monotherapy. Treg infusion resulted in a long-lasting dose-dependent increase in peripheral blood Tregs together with an increase in marginal zone B cell numbers. We identified a pretransplantation immune phenotype suggesting a high risk of unsuccessful ex-vivo Treg expansion. Autologous Treg therapy is feasible, safe, and is potentially associated with a lower rejection rate than standard immunosuppression. Treg therapy may provide an exciting opportunity to minimize immunosuppression therapy and improve long-term outcomes.
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http://dx.doi.org/10.1111/ajt.16395DOI Listing
April 2021

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

Single-cell mass cytometry of microglia in major depressive disorder reveals a non-inflammatory phenotype with increased homeostatic marker expression.

Transl Psychiatry 2020 09 11;10(1):310. Epub 2020 Sep 11.

Department of Neuropsychiatry and Laboratory of Molecular Psychiatry, Charité - Universitätsmedizin Berlin, Berlin, Germany.

Stress-induced disturbances of brain homeostasis and neuroinflammation have been implicated in the pathophysiology of mood disorders. In major depressive disorder (MDD), elevated levels of proinflammatory cytokines and chemokines can be found in peripheral blood, but very little is known about the changes that occur directly in the brain. Microglia are the primary immune effector cells of the central nervous system and exquisitely sensitive to changes in the brain microenvironment. Here, we performed the first single-cell analysis of microglia from four different post-mortem brain regions (frontal lobe, temporal lobe, thalamus, and subventricular zone) of medicated individuals with MDD compared to controls. We found no evidence for the induction of inflammation-associated molecules, such as CD11b, CD45, CCL2, IL-1β, IL-6, TNF, MIP-1β (CCL4), IL-10, and even decreased expression of HLA-DR and CD68 in microglia from MDD cases. In contrast, we detected increased levels of the homeostatic proteins P2Y receptor, TMEM119 and CCR5 (CD195) in microglia from all brain regions of individuals with MDD. We also identified enrichment of non-inflammatory CD206 macrophages in the brains of MDD cases. In sum, our results suggest enhanced homeostatic functions of microglia in MDD.
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http://dx.doi.org/10.1038/s41398-020-00992-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7486938PMC
September 2020

Single-cell mass cytometry reveals complex myeloid cell composition in active lesions of progressive multiple sclerosis.

Acta Neuropathol Commun 2020 08 18;8(1):136. Epub 2020 Aug 18.

Department of Neuropsychiatry and Laboratory of Molecular Psychiatry, Charité - Universitätsmedizin Berlin, Berlin, Germany.

Myeloid cells contribute to inflammation and demyelination in the early stages of multiple sclerosis (MS), but it is still unclear to what extent these cells are involved in active lesion formation in progressive MS (PMS). Here, we have harnessed the power of single-cell mass cytometry (CyTOF) to compare myeloid cell phenotypes in active lesions of PMS donors with those in normal-appearing white matter from the same donors and control white matter from non-MS donors. CyTOF measurements of a total of 74 targeted proteins revealed a decreased abundance of homeostatic and TNF microglia, and an increase in highly phagocytic and activated microglia states in active lesions of PMS donors. Interestingly, in contrast to results obtained from studies of the inflammatory early disease stages of MS, infiltrating monocyte-derived macrophages were scarce in active lesions of PMS, suggesting fundamental differences of myeloid cell composition in advanced stages of PMS.
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http://dx.doi.org/10.1186/s40478-020-01010-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7437178PMC
August 2020

Severe COVID-19 Is Marked by a Dysregulated Myeloid Cell Compartment.

Cell 2020 09 5;182(6):1419-1440.e23. Epub 2020 Aug 5.

Department of Infectious Diseases and Respiratory Medicine, Charité, Universitätsmedizin Berlin, Berlin, Germany; German Center for Lung Research (DZL).

Coronavirus disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progress to severe disease and respiratory failure. The mechanism of protective immunity in mild forms and the pathogenesis of severe COVID-19 associated with increased neutrophil counts and dysregulated immune responses remain unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole-blood and peripheral-blood mononuclear cells to determine changes in immune cell composition and activation in mild versus severe COVID-19 (242 samples from 109 individuals) over time. HLA-DRCD11c inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DR monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in the myeloid cell compartment associated with severe COVID-19.
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http://dx.doi.org/10.1016/j.cell.2020.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7405822PMC
September 2020

The H-Y Antigen in Embryonic Stem Cells Causes Rejection in Syngeneic Female Recipients.

Stem Cells Dev 2020 09 25;29(18):1179-1189. Epub 2020 Aug 25.

Transplant and Stem Cell Immunobiology Lab, Department of Surgery, University of California, San Francisco, California, USA.

Pluripotent stem cells are promising candidates for cell-based regenerative therapies. To avoid rejection of transplanted cells, several approaches are being pursued to reduce immunogenicity of the cells or modulate the recipient's immune response. These include gene editing to reduce the antigenicity of cell products, immunosuppression of the host, or using major histocompatibility complex-matched cells from cell banks. In this context, we have investigated the antigenicity of H-Y antigens, a class of minor histocompatibility antigens encoded by the Y chromosome, to assess whether the gender of the donor affects the cell's antigenicity. In a murine transplant model, we show that the H-Y antigen in undifferentiated embryonic stem cells (ESCs), as well as ESC-derived endothelial cells, provokes T- and B cell responses in female recipients.
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http://dx.doi.org/10.1089/scd.2019.0299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7482111PMC
September 2020

Regulatory cell therapy in kidney transplantation (The ONE Study): a harmonised design and analysis of seven non-randomised, single-arm, phase 1/2A trials.

Lancet 2020 05;395(10237):1627-1639

Department of Surgery, University of Regensburg, University Hospital Regensburg, Regensburg, Germany; Division of Personalized Tumor Therapy, Fraunhofer Institute for Experimental Medicine and Toxicology, Regensburg, Germany; Regensburg Center for Interventional Immunology, University of Regensburg, Regensburg, Germany. Electronic address:

Background: Use of cell-based medicinal products (CBMPs) represents a state-of-the-art approach for reducing general immunosuppression in organ transplantation. We tested multiple regulatory CBMPs in kidney transplant trials to establish the safety of regulatory CBMPs when combined with reduced immunosuppressive treatment.

Methods: The ONE Study consisted of seven investigator-led, single-arm trials done internationally at eight hospitals in France, Germany, Italy, the UK, and the USA (60 week follow-up). Included patients were living-donor kidney transplant recipients aged 18 years and older. The reference group trial (RGT) was a standard-of-care group given basiliximab, tapered steroids, mycophenolate mofetil, and tacrolimus. Six non-randomised phase 1/2A cell therapy group (CTG) trials were pooled and analysed, in which patients received one of six CBMPs containing regulatory T cells, dendritic cells, or macrophages; patient selection and immunosuppression mirrored the RGT, except basiliximab induction was substituted with CBMPs and mycophenolate mofetil tapering was allowed. None of the trials were randomised and none of the individuals involved were masked. The primary endpoint was biopsy-confirmed acute rejection (BCAR) within 60 weeks after transplantation; adverse event coding was centralised. The RTG and CTG trials are registered with ClinicalTrials.gov, NCT01656135, NCT02252055, NCT02085629, NCT02244801, NCT02371434, NCT02129881, and NCT02091232.

Findings: The seven trials took place between Dec 11, 2012, and Nov 14, 2018. Of 782 patients assessed for eligibility, 130 (17%) patients were enrolled and 104 were treated and included in the analysis. The 66 patients who were treated in the RGT were 73% male and had a median age of 47 years. The 38 patients who were treated across six CTG trials were 71% male and had a median age of 45 years. Standard-of-care immunosuppression in the recipients in the RGT resulted in a 12% BCAR rate (expected range 3·2-18·0). The overall BCAR rate for the six parallel CTG trials was 16%. 15 (40%) patients given CBMPs were successfully weaned from mycophenolate mofetil and maintained on tacrolimus monotherapy. Combined adverse event data and BCAR episodes from all six CTG trials revealed no safety concerns when compared with the RGT. Fewer episodes of infections were registered in CTG trials versus the RGT.

Interpretation: Regulatory cell therapy is achievable and safe in living-donor kidney transplant recipients, and is associated with fewer infectious complications, but similar rejection rates in the first year. Therefore, immune cell therapy is a potentially useful therapeutic approach in recipients of kidney transplant to minimise the burden of general immunosuppression.

Funding: The 7th EU Framework Programme.
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http://dx.doi.org/10.1016/S0140-6736(20)30167-7DOI Listing
May 2020

Virus-associated anterior uveitis and secondary glaucoma: Diagnostics, clinical characteristics, and surgical options.

PLoS One 2020 24;15(2):e0229260. Epub 2020 Feb 24.

Berlin Institute of Health, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin, Germany.

In this retrospective, single-center, observational study, we compared the clinical characteristics, analyzed the glaucoma development, and the glaucoma surgery requirement mediators in patients with different virus-associated anterior uveitis (VAU). In total, 270 patients (= eyes) with VAU confirmed by positive Goldmann-Witmer coefficients (GWC) for cytomegalovirus (CMV), herpes simplex virus (HSV), varicella-zoster virus (VZV), rubella virus (RV), and multiple virus (MV) were included. Clinical records of these patients were analyzed. Demographic constitution, clinical findings, glaucoma development, and surgeries were recorded. The concentrations of 27 immune mediators were measured in 150 samples of aqueous humor. The GWC analysis demonstrated positive results for CMV in 57 (21%), HSV in 77 (29%), VZV in 45 (17%), RV in 77 (29%), and MV in 14 (5%) patients. CMV and RV AU occurred predominantly in younger and male patients, while VZV and HSV AU appeared mainly with the elderly and females (P<0.0001). The clinical features of all viruses revealed many similarities. In total, 52 patients (19%) showed glaucomatous damage and of these, 27 patients (10%) needed a glaucoma surgery. Minimal-invasive glaucoma surgery (MIGS) showed a reliable IOP reduction in the short-term period. In 10 patients (37%), the first surgical intervention failed and a follow-up surgery was required. We conclude that different virus entities in anterior uveitis present specific risks for the development of glaucoma as well as necessary surgery. MIGS can be suggested as first-line-treatment in individual cases, however, the device needs to be carefully chosen by experienced specialists based on the individual needs of the patient. Filtrating glaucoma surgery can be recommended in VAU as an effective therapy to reduce the IOP over a longer period of time.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0229260PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7039515PMC
June 2020

Multi-parameter immune profiling of peripheral blood mononuclear cells by multiplexed single-cell mass cytometry in patients with early multiple sclerosis.

Sci Rep 2019 12 19;9(1):19471. Epub 2019 Dec 19.

Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.

Multiple sclerosis (MS) is an inflammatory demyelinating and neurodegenerative disease of the central nervous system (CNS). Studies in rodent models demonstrated an association of CNS-infiltrating monocyte-derived macrophages with disease severity. However, little is known about humans. Here, we performed an exploratory analysis of peripheral blood mononuclear cells (PBMCs) isolated from healthy controls and drug-naïve patients with early MS using multiplexed single-cell mass cytometry and algorithm-based data analysis. Two antibody panels comprising a total of 64 antibodies were designed to comprehensively analyse diverse immune cell populations, with particular emphasis on monocytes. PBMC composition and marker expression were overall similar between the groups. However, an increased abundance of CCR7 and IL-6 T cells was detected in early MS-PBMCs, whereas NFAT1T-betCD4 T cells were decreased. Similarly, we detected changes in the subset composition of the CCR7 and MIPβ HLA-DR lymphocyte compartment. Only mild alterations were detected in monocytes/myeloid cells of patients with early MS, namely a decreased abundance of CD141IRF8CXCR3CD68 dendritic cells. Unlike in Crohn's disease, no significant differences were found in the monocyte fraction of patients with early MS compared to healthy controls. This study provides a valuable resource for future studies designed to characterise and target diverse PBMC subsets in MS.
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http://dx.doi.org/10.1038/s41598-019-55852-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6923404PMC
December 2019

Multi-Parameter Analysis of Biobanked Human Bone Marrow Stromal Cells Shows Little Influence for Donor Age and Mild Comorbidities on Phenotypic and Functional Properties.

Front Immunol 2019 8;10:2474. Epub 2019 Nov 8.

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

Heterogeneous populations of human bone marrow-derived stromal cells (BMSC) are among the most frequently tested cellular therapeutics for treating degenerative and immune disorders, which occur predominantly in the aging population. Currently, it is unclear whether advanced donor age and commonly associated comorbidities affect the properties of -expanded BMSCs. Thus, we stratified cells from adult and elderly donors from our biobank ( = 10 and = 13, mean age 38 and 72 years, respectively) and compared their phenotypic and functional performance, using multiple assays typically employed as minimal criteria for defining multipotent mesenchymal stromal cells (MSCs). We found that BMSCs from both cohorts meet the standard criteria for MSC, exhibiting similar morphology, growth kinetics, gene expression profiles, and pro-angiogenic and immunosuppressive potential and the capacity to differentiate toward adipogenic, chondrogenic, and osteogenic lineages. We found no substantial differences between cells from the adult and elderly cohorts. As positive controls, we studied the impact of aging and inflammatory cytokine stimulation. Both conditions clearly affected the cellular properties, independent of donor age. We conclude that aging rather than donor aging influences BMSC characteristics.
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http://dx.doi.org/10.3389/fimmu.2019.02474DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6857652PMC
October 2020

Long-Term Signs of T Cell and Myeloid Cell Activation After Intestinal Transplantation With Cellular Rejections Contributing to Further Increase of CD16 Cell Subsets.

Front Immunol 2019 7;10:866. Epub 2019 May 7.

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

The intestine mediates a delicate balance between tolerogenic and inflammatory immune responses. The continuous pathogen encounter might also augment immune cell responses contributing to complications observed upon intestinal transplantation (ITx). We thus hypothesized that ITx patients show persistent signs of immune cell activation affecting both the adaptive and innate immune cell compartment. Information on the impact of intestinal grafts on immune cell composition, however, especially in the long-term is sparse. We here assessed activated and differentiated adaptive and innate immune subsets according to time, previous experience of cellular or antibody-mediated rejections or type of transplant after ITx applying multi-parametric flow cytometry, gene expression, serum cytokine and chemokine profiling. ITx patients showed an increase in CD16 expressing monocytes and myeloid dendritic cells (DCs) compared to healthy controls. This was even detectable in patients who were transplanted more than 10 years ago. Also, conventional CD4 and CD8 T cells showed persistent signs of activation counterbalanced by increased activated CCR4 regulatory T cells. Patients with previous cellular rejections had even higher proportions of CD16 monocytes and DCs, whereas transplanting higher donor mass with multi-visceral grafts was associated with increased T cell activation. The persistent inflammation and innate immune cell activation might contribute to unsatisfactory results after ITx.
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http://dx.doi.org/10.3389/fimmu.2019.00866DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6514047PMC
September 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

Immune Mediator Profile in Aqueous Humor Differs in Patients with Primary Acquired Ocular Toxoplasmosis and Recurrent Acute Ocular Toxoplasmosis.

Mediators Inflamm 2019 17;2019:9356728. Epub 2019 Feb 17.

Department of Ophthalmology, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Germany.

Purpose: To compare the intraocular cytokine and chemokine profiles in patients with acute primary acquired ocular toxoplasmosis (pOT) or recurrent ocular toxoplasmosis (rOT) and to correlate them with their clinical characteristics.

Methods: Aqueous humor samples were collected from 62 consecutive patients (21 pOT, 30 rOT, and 11 noninfected controls) and analyzed by multiplex assay. Correlations were assessed between cytokine/chemokine levels, type of inflammatory response (T1, T2, and T17), and clinical characteristics. In all OT patients, the clinical diagnosis of either pOT or rOT was confirmed by positive intraocular Goldmann/Witmer-Desmonts coefficient. Correlations were assessed between a preselected panel of immune mediators and the clinical characteristics of OT.

Results: In pOT patients, increased levels of IL-2, IFN-, TNF-, IL-15, IL-4, IL-5, IL-9, IL-13, IL-17, IL-1R, IL-6, IL-1, and chemokines MIP-1, MIP-1, IP-10, Eotaxin, IL-8, RANTES, PDGF-bb, GM-CSF, G-CSF, and MCP-1 were found in comparison to those in controls ( < 0.05). Patients with rOT showed elevated levels of IL-2, IFN-, TNF-, IL-15, IL-4, IL-5, IL-9, IL-17, IL-1R, IL-6, IL-1, and chemokines MIP-1, IP-10, Eotaxin, IL-8, RANTES, PDGF-bb, G-CSF, and MCP-1 compared to controls ( < 0.05). In addition, IL-7 ( = 0.028) differed between pOT and rOT; IL-9 ( = 0.054) and IL-13 ( = 0.051) showed a tendency of higher concentration in pOT than in rOT. A negative correlation was found between IL-7 ( = 0.017) as well as IL-9 ( = 0.008) and the number of recurrences. Cytokine ratios showed no difference between pOT and rOT, indicating a dominant T1-type response in both infectious groups. Moreover, a positive correlation was detected between IL-7, VEGF, IL-13 and age at aqueous humor sampling ( < 0.05).

Conclusions: This study for the first time shows subtle differences between the intraocular cytokine profiles in patients with either acute pOT or rOT.
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http://dx.doi.org/10.1155/2019/9356728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398019PMC
July 2019

Standardized assay for assessment of minimal residual disease in blood, bone marrow and apheresis from patients with plasma cell myeloma.

Sci Rep 2019 02 27;9(1):2922. Epub 2019 Feb 27.

Charité Stem Cell Facility, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Augustenburger Platz 1, Berlin, 13353, Germany.

The recent advances in myeloma treatment result in significantly better outcomes, defined as increased progression free survival (PFS) and overall survival (OS). Since there is a proven correlation between the extend of response and prolonged survival, there is an urgent need for highly sensitive assays for the detection of minimal residual disease (MRD). Next generation flow cytometry has become a valuable approach for sensitive evaluation of the depth of complete response (CR). Here, we report the diagnostic performance and validation results of a single-tube 9-color panel assay. The validation design included intra-assay analysis measuring accuracy, inter-assay analysis estimating method's linearity and precision and inter-assay analysis evaluating repeatability. Furthermore, in inter-operator analysis assessed the comparability of the result analysis of different operators. Staining stability was evaluated in age-of-stain experiments. Our validation results show that a reliable detection of residual myeloma cells is feasible to a detection level of 10 with a single-tube assay for a variety of materials (peripheral blood, bone marrow and stem cell apheresis). This study establishes highly sensitive, fully standardized approach for MRD detection in myeloma that is ready for implementation in routine diagnostic laboratories.
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http://dx.doi.org/10.1038/s41598-019-39631-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6393516PMC
February 2019

The Value of a Rapid Test of Human Regulatory T Cell Function Needs to be Revised.

Front Immunol 2019 5;10:150. Epub 2019 Feb 5.

Institute for Medical Immunology, Charité-Universitätsmedizin Berlin, Berlin, Germany.

CD4CD25FoxP3 human regulatory T (T) are promising candidates for reshaping undesired immunity/inflammation by adoptive cell transfer, yet their application is strongly dependent on robust assays testing their functionality. Several studies along with first clinical data indicate T to be auspicious to use for future cell therapies, e.g., to induce tolerance after solid organ transplantation. To this end, T suppressive capacity has to be thoroughly evaluated prior to any therapeutic application. A 7 h-protocol for the assessment of T function by suppression of the early activation markers CD154 and CD69 on CD4CD25 responder T (T) upon polyclonal stimulation via αCD3/28-coated activating microbeads has previously been published. Even though this assay has since been applied by various groups, the protocol comes with a critical pitfall, which is yet not corrected by the journal of its original publication. Our results demonstrate that the observed decrease in activation marker frequency on T is due to competition for αCD3/28-coated microbeads as opposed to a T-attributable effect and therefore the protocol cannot further be used as a diagnostic test to assess suppressive T function.
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http://dx.doi.org/10.3389/fimmu.2019.00150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6370705PMC
January 2020

Human microglia regional heterogeneity and phenotypes determined by multiplexed single-cell mass cytometry.

Nat Neurosci 2019 01 17;22(1):78-90. Epub 2018 Dec 17.

Department of Neuropsychiatry and Laboratory of Molecular Psychiatry, Charité-Universitätsmedizin Berlin, Berlin, Germany.

Microglia, the specialized innate immune cells of the CNS, play crucial roles in neural development and function. Different phenotypes and functions have been ascribed to rodent microglia, but little is known about human microglia (huMG) heterogeneity. Difficulties in procuring huMG and their susceptibility to cryopreservation damage have limited large-scale studies. Here we applied multiplexed mass cytometry for a comprehensive characterization of postmortem huMG (10 - 10 cells). We determined expression levels of 57 markers on huMG isolated from up to five different brain regions of nine donors. We identified the phenotypic signature of huMG, which was distinct from peripheral myeloid cells but was comparable to fresh huMG. We detected microglia regional heterogeneity using a hybrid workflow combining Cytobank and R/Bioconductor for multidimensional data analysis. Together, these methodologies allowed us to perform high-dimensional, large-scale immunophenotyping of huMG at the single-cell level, which facilitates their unambiguous profiling in health and disease.
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http://dx.doi.org/10.1038/s41593-018-0290-2DOI Listing
January 2019

A standardized immune phenotyping and automated data analysis platform for multicenter biomarker studies.

JCI Insight 2018 12 6;3(23). Epub 2018 Dec 6.

Department of Surgery, University of British Columbia, Vancouver, British Columbia, Canada.

The analysis and validation of flow cytometry-based biomarkers in clinical studies are limited by the lack of standardized protocols that are reproducible across multiple centers and suitable for use with either unfractionated blood or cryopreserved PBMCs. Here we report the development of a platform that standardizes a set of flow cytometry panels across multiple centers, with high reproducibility in blood or PBMCs from either healthy subjects or patients 100 days after hematopoietic stem cell transplantation. Inter-center comparisons of replicate samples showed low variation, with interindividual variation exceeding inter-center variation for most populations (coefficients of variability <20% and interclass correlation coefficients >0.75). Exceptions included low-abundance populations defined by markers with indistinct expression boundaries (e.g., plasmablasts, monocyte subsets) or populations defined by markers sensitive to cryopreservation, such as CD62L and CD45RA. Automated gating pipelines were developed and validated on an independent data set, revealing high Spearman's correlations (rs >0.9) with manual analyses. This workflow, which includes pre-formatted antibody cocktails, standardized protocols for acquisition, and validated automated analysis pipelines, can be readily implemented in multicenter clinical trials. This approach facilitates the collection of robust immune phenotyping data and comparison of data from independent studies.
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http://dx.doi.org/10.1172/jci.insight.121867DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6328091PMC
December 2018

Different composition of intraocular immune mediators in Posner-Schlossman-Syndrome and Fuchs' Uveitis.

PLoS One 2018 26;13(6):e0199301. Epub 2018 Jun 26.

Department of Ophthalmology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.

Posner-Schlossman-Syndrome (PSS) is clinically characterized by acute, recurrent, mild, unilateral uveitis anterior accompanied by elevated intraocular pressure (IOP). Fuchs´ Uveitis (FU) is a chronic, low-grade-inflammatory disorder, involving anterior uvea and vitreous. The clinical findings show remarkable similarities as well as differences. In our study, we determine the composition of immune mediators in aqueous humor of patients with PSS and FU and evaluate if immune mediators play a crucial role in specific viral intraocular inflammation and IOP rises. Aqueous humor samples from 81 uveitis patients (= eyes) presenting with either PSS or FU were collected at one time point. Local intraocular antibody synthesis to rubella virus was confirmed in 65 patients, whereas 16 were tested positively for human cytomegalovirus. Thirteen patients with PSS and 10 patients with FU were treated with glaucoma medications. Additionally, 11 cataract patients acted as control group. Immune mediator concentrations were measured by Bio-Plex Pro assay. We observed in both PSS (IFN-γ: 174.9 pg/mL; TNF-α: 25.1 pg/mL) and FU (IFN-γ: 25.4 pg/mL; TNF-α: 27.2 pg/mL) groups a significantly increased level of T-helper 1 immune mediators compared to controls (IFN-γ, TNF-α: 0 pg/mL) [median]. Notably, PSS patients (IL-1RA: 73.4 pg/mL; IL-8: 199.4 pg/mL; IL-10: 33.4 pg/mL; IP-10: 126350 pg/mL) showed a stronger and more active ocular inflammatory response, than FU patients (IL-1RA: 4.3 pg/mL; IL-8: 72.4 pg/mL; IL-10: 1.6 pg/mL; IP-10: 57400 pg/mL). Furthermore, a negative correlation between mediators and IOP was seen in the PSS group, potentially caused by acetazolamide-treatment. Our findings show that immune mediators play a crucial role in specific viral intraocular inflammation and influence IOP levels. Remarkable similarities but also significant differences of immune mediator concentrations are apparent in PSS compared to FU. High concentrations of IL-1RA, IL-8, IL-10, and IP-10 correlate with active inflammation in PSS, while FU may trigger chronic inflammation. Our data also substantiated a very similar composition of cytokines in those patients from the PSS group suffering from ocular hypertension and thus offers a potential explanation model for a negative correlation between mediators and IOP.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0199301PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6019249PMC
April 2019

CD96 expression determines the inflammatory potential of IL-9-producing Th9 cells.

Proc Natl Acad Sci U S A 2018 03 12;115(13):E2940-E2949. Epub 2018 Mar 12.

Charité-Universitaetsmedizin Berlin, corporate member of Freie Universitaet Berlin, Humboldt-Universitaet zu Berlin, and Berlin Institute of Health, Institute for Medical Immunology, 13353 Berlin, Germany;

Recent findings demonstrated proinflammatory functions of interleukin (IL)-9-producing T helper type (Th) 9 cells in the pathogenesis of intestinal bowel diseases (IBDs). However, also antiinflammatory properties have been ascribed to Th9 cells, pointing to a functional heterogeneity. To dissect the specific expression pattern and, especially, diversity of murine antigen-specific Th9 cells, we applied single cell transcription profiling. Th9 cells displayed reduced expression of typical activation markers, such as and , whereas expression of and was increased compared with other Th subsets. Importantly, we identified two subsets of Th9 cells differing above all in their CD96 expression. The heterogeneous CD96 expression was specific for Th9 cells and not observed for other Th subtypes, such as Th1 cells. Lower CD96 expression was also observed in human IL-9 compared with IFN-γ T cells. Although was highly transcribed by all Th9 cells, IL-9 mRNA and protein expression was increased in CD96 cells. Transfer of CD96 Th9 cells into recombination activating gene 1-deficient ( ) mice caused severe weight loss, intestinal and colonic inflammation, and destruction of allogeneic skin grafts and thus showed high inflammatory potential. This was associated with their expansion and tissue accumulation. Contrastingly, CD96 Th9 cells did not cause colitis and showed reduced expansion and migratory potential. Blockade of CD96 completely restored the expansion and inflammatory properties of CD96 Th9 cells. Collectively, our data suggest an inhibitory role for the cosignaling receptor CD96 in Th9 cells, raising new opportunities in the treatment of IL-9-associated inflammations such as IBD.
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http://dx.doi.org/10.1073/pnas.1708329115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5879650PMC
March 2018

Epigenomic Profiling of Human CD4 T Cells Supports a Linear Differentiation Model and Highlights Molecular Regulators of Memory Development.

Immunity 2016 11;45(5):1148-1161

Experimental Rheumatology, German Rheumatism Research Centre, 10117 Berlin, Germany. Electronic address:

The impact of epigenetics on the differentiation of memory T (Tmem) cells is poorly defined. We generated deep epigenomes comprising genome-wide profiles of DNA methylation, histone modifications, DNA accessibility, and coding and non-coding RNA expression in naive, central-, effector-, and terminally differentiated CD45RA CD4 Tmem cells from blood and CD69 Tmem cells from bone marrow (BM-Tmem). We observed a progressive and proliferation-associated global loss of DNA methylation in heterochromatic parts of the genome during Tmem cell differentiation. Furthermore, distinct gradually changing signatures in the epigenome and the transcriptome supported a linear model of memory development in circulating T cells, while tissue-resident BM-Tmem branched off with a unique epigenetic profile. Integrative analyses identified candidate master regulators of Tmem cell differentiation, including the transcription factor FOXP1. This study highlights the importance of epigenomic changes for Tmem cell biology and demonstrates the value of epigenetic data for the identification of lineage regulators.
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http://dx.doi.org/10.1016/j.immuni.2016.10.022DOI Listing
November 2016

Wild immunology assessed by multidimensional mass cytometry.

Cytometry A 2017 01 12;91(1):85-95. Epub 2016 Jul 12.

Regenerative Immunology and Aging, BCRT, Charité Universitätsmedizin Berlin, Berlin, Germany.

A great part of our knowledge on mammalian immunology has been established in laboratory settings. The use of inbred mouse strains enabled controlled studies of immune cell and molecule functions in defined settings. These studies were usually performed in specific-pathogen free (SPF) environments providing standardized conditions. In contrast, mammalians including humans living in their natural habitat are continuously facing pathogen encounters throughout their life. The influences of environmental conditions on the signatures of the immune system and on experimental outcomes are yet not well defined. Thus, the transferability of results obtained in current experimental systems to the physiological human situation has always been a matter of debate. Studies elucidating the diversity of "wild immunology" imprintings in detail and comparing it with those of "clean" lab mice are sparse. Here, we applied multidimensional mass cytometry to dissect phenotypic and functional differences between distinct groups of laboratory and pet shop mice as a source for "wild mice". For this purpose, we developed a 31-antibody panel for murine leukocyte subsets identification and a 35-antibody panel assessing various cytokines. Established murine leukocyte populations were easily identified and diverse immune signatures indicative of numerous pathogen encounters were classified particularly in pet shop mice and to a lesser extent in quarantine and non-SPF mice as compared to SPF mice. In addition, unsupervised analysis identified distinct clusters that associated strongly with the degree of pathogenic priming, including increased frequencies of activated NK cells and antigen-experienced B- and T-cell subsets. Our study unravels the complexity of immune signatures altered under physiological pathogen challenges and highlights the importance of carefully adapting laboratory settings for immunological studies in mice, including drug and therapy testing. © 2016 International Society for Advancement of Cytometry.
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http://dx.doi.org/10.1002/cyto.a.22906DOI Listing
January 2017

Age and gender leucocytes variances and references values generated using the standardized ONE-Study protocol.

Cytometry A 2016 06 3;89(6):543-64. Epub 2016 May 3.

Institute of Medical Immunology, Charité Universitätsmedizin Berlin, Germany.

Flow cytometry is now accepted as an ideal technology to reveal changes in immune cell composition and function. However, it is also an error-prone and variable technology, which makes it difficult to reproduce findings across laboratories. We have recently developed a strategy to standardize whole blood flow cytometry. The performance of our protocols was challenged here by profiling samples from healthy volunteers to reveal age- and gender-dependent differences and to establish a standardized reference cohort for use in clinical trials. Whole blood samples from two different cohorts were analyzed (first cohort: n = 52, second cohort: n = 46, both 20-84 years with equal gender distribution). The second cohort was run as a validation cohort by a different operator. The "ONE Study" panels were applied to analyze expression of >30 different surface markers to enumerate proportional and absolute numbers of >50 leucocyte subsets. Indeed, analysis of the first cohort revealed significant age-dependent changes in subsets e.g. increased activated and differentiated CD4(+) and CD8(+) T cell subsets, acquisition of a memory phenotype for Tregs as well as decreased MDC2 and Marginal Zone B cells. Males and females showed different dynamics in age-dependent T cell activation and differentiation, indicating faster immunosenescence in males. Importantly, although both cohorts consisted of a small sample size, our standardized approach enabled validation of age-dependent changes with the second cohort. Thus, we have proven the utility of our strategy and generated reproducible reference ranges accounting for age- and gender-dependent differences, which are crucial for a better patient monitoring and individualized therapy. © 2016 International Society for Advancement of Cytometry.
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http://dx.doi.org/10.1002/cyto.a.22855DOI Listing
June 2016

Standardized Multi-Color Flow Cytometry and Computational Biomarker Discovery.

Methods Mol Biol 2016 ;1371:225-38

Institute of Medical Immunology, Charité University Medicine, Berlin, 10117, Germany.

Multi-color flow cytometry has become a valuable and highly informative tool for diagnosis and therapeutic monitoring of patients with immune deficiencies or inflammatory disorders. However, the method complexity and error-prone conventional manual data analysis often result in a high variability between different analysts and research laboratories. Here, we provide strategies and guidelines aiming at a more standardized multi-color flow cytometric staining and unsupervised data analysis for whole blood patient samples.
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http://dx.doi.org/10.1007/978-1-4939-3139-2_15DOI Listing
August 2016

TCAIM decreases T cell priming capacity of dendritic cells by inhibiting TLR-induced Ca2+ influx and IL-2 production.

J Immunol 2015 Apr 6;194(7):3136-46. Epub 2015 Mar 6.

Institute of Medical Immunology, Charite University Medicine, Berlin 13353, Germany; Berlin Brandenburg Center for Regenerative Therapies, Charite University Medicine, Berlin 13353, Germany;

We previously showed that the T cell activation inhibitor, mitochondrial (Tcaim) is highly expressed in grafts of tolerance-developing transplant recipients and that the encoded protein is localized within mitochondria. In this study, we show that CD11c(+) dendritic cells (DCs), as main producers of TCAIM, downregulate Tcaim expression after LPS stimulation or in vivo alloantigen challenge. LPS-stimulated TCAIM-overexpressing bone marrow-derived DC (BMDCs) have a reduced capacity to induce proliferation of and cytokine expression by cocultured allogeneic T cells; this is not due to diminished upregulation of MHC or costimulatory molecules. Transcriptional profiling also revealed normal LPS-mediated upregulation of the majority of genes involved in TLR signaling. However, TCAIM BMDCs did not induce Il2 mRNA expression upon LPS stimulation in comparison with Control-BMDCs. In addition, TCAIM overexpression abolished LPS-mediated Ca(2+) influx and mitochondrial reactive oxygen species formation. Addition of IL-2 to BMDC-T cell cocultures restored the priming capacity of TCAIM BMDCs for cocultured allogeneic CD8(+) T cells. Furthermore, BMDCs of IL-2-deficient mice showed similarly abolished LPS-induced T cell priming as TCAIM-overexpressing wild type BMDCs. Thus, TCAIM interferes with TLR4 signaling in BMDCs and subsequently impairs their T cell priming capacity, which supports its role for tolerance induction.
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http://dx.doi.org/10.4049/jimmunol.1400713DOI Listing
April 2015

The use of novel diagnostics to individualize immunosuppression following transplantation.

Transpl Int 2015 Aug 4;28(8):911-20. Epub 2015 Feb 4.

Institute of Medical Immunology, CCM, Charité University Berlin, Berlin, Germany.

Despite major improvements in short-term survival of organ allografts, long-term graft survival has not changed significantly. It is also known that toxic side effects of current immunosuppressive drugs (IS) especially calcineurin inhibitors (CNI) contribute to the unsatisfactory graft and patient survival following transplantation. Thus, clinicians strive to reduce or wean IS in potentially eligible patients. Research in the last 10 years has focussed on identification of biomarkers suitable for patient stratification in minimization or weaning trials. Most of the described biomarkers have been run retrospectively on samples collected within single-centre trials. Thus, often their performance has not been validated in other potentially multicentre clinical trials. Ultimately, the utility of biomarkers to identify potential weaning candidates should be investigated in large randomized prospective trials. In particular, for testing in such trials, we need more information about the accuracy, reproducibility, stability and limitations of the described biomarkers. Also, data repositories summarizing crucial information on biomarker performance in age- and gender-matched healthy individuals of different ethnicity are missing. This together with improved bioinformatics tools might help in developing better scores for patient stratification. Here, we will summarize the current results, knowledge and limitations on biomarkers for drug minimization or weaning trials.
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http://dx.doi.org/10.1111/tri.12527DOI Listing
August 2015

Standardization of whole blood immune phenotype monitoring for clinical trials: panels and methods from the ONE study.

Transplant Res 2013 Oct 25;2(1):17. Epub 2013 Oct 25.

Institute of Medical Immunology, Charité - Universitätsmedizin Berlin, Augustenburger Platz 1, Berlin 13353, Germany.

Background: Immune monitoring by flow cytometry is a fast and highly informative way of studying the effects of novel therapeutics aimed at reducing transplant rejection or treating autoimmune diseases. The ONE Study consortium has recently initiated a series of clinical trials aimed at using different cell therapies to promote tolerance to renal allografts. To compare the effectiveness of different cell therapies, the consortium developed a robust immune monitoring strategy, including procedures for whole blood (WB) leukocyte subset profiling by flow cytometry.

Methods: Six leukocyte profiling panels computing 7- to 9-surface marker antigens for monitoring the major leukocyte subsets as well as characteristics of T cell, B cell, and dendritic cell (DC) subsets were designed. The precision and variability of these panels were estimated. The assay was standardized within eight international laboratories using Flow-Set Pro beads for mean fluorescence intensity target definition and the flow cytometer setup procedure. Standardization was demonstrated by performing inter-site comparisons.

Results: Optimized methods for sample collection, storage, preparation, and analysis were established, including protocols for gating target subsets. WB specimen age testing demonstrated that staining must be performed within 4 hours of sample collection to keep variability low, meaning less than or equal to 10% for the majority of defined leukocyte subsets. Inter-site comparisons between all participating centers testing shipped normal WB revealed good precision, with a variability of 0.05% to 30% between sites. Intra-assay analyses revealed a variability of 0.05% to 20% for the majority of subpopulations. This was dependent on the frequency of the particular subset, with smaller subsets showing higher variability. The intra-assay variability performance defined limits of quantitation (LoQ) for subsets, which will be the basis for assessing statistically significant differences achieved by the different cell therapies.

Conclusions: Local performance and central analysis of the ONE Study flow cytometry panel yields acceptable variability in a standardized assay at multiple international sites. These panels and procedures with WB allow unmanipulated analysis of changes in absolute cell numbers of leukocyte subsets in single- or multicenter clinical trials. Accordingly, we propose the ONE Study panel may be adopted as a standardized method for monitoring patients in clinical trials enrolling transplant patients, particularly trials of novel tolerance promoting therapies, to facilitate fair and meaningful comparisons between trials.
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http://dx.doi.org/10.1186/2047-1440-2-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3827923PMC
October 2013

Generation of highly effective and stable murine alloreactive Treg cells by combined anti-CD4 mAb, TGF-β, and RA treatment.

Eur J Immunol 2013 Dec 9;43(12):3291-305. Epub 2013 Sep 9.

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

The transfer of alloreactive regulatory T (aTreg) cells into transplant recipients represents an attractive treatment option to improve long-term graft acceptance. We recently described a protocol for the generation of aTreg cells in mice using a nondepleting anti-CD4 antibody (aCD4). Here, we investigated whether adding TGF-β and retinoic acid (RA) or rapamycin (Rapa) can further improve aTreg-cell generation and function. Murine CD4(+) T cells were cultured with allogeneic B cells in the presence of aCD4 alone, aCD4+TGF-β+RA or aCD4+Rapa. Addition of TGF-β+RA or Rapa resulted in an increase of CD25(+)Foxp3(+)-expressing T cells. Expression of CD40L and production of IFN-γ and IL-17 was abolished in aCD4+TGF-β+RA aTreg cells. Additionally, aCD4+TGF-β+RA aTreg cells showed the highest level of Helios and Neuropilin-1 co-expression. Although CD25(+)Foxp3(+) cells from all culture conditions displayed complete demethylation of the Treg-specific demethylated region, aCD4+TGF-β+RA Treg cells showed the most stable Foxp3 expression upon restimulation. Consequently, aCD4+TGF-β+RA aTreg cells suppressed effector T-cell differentiation more effectively in comparison to aTreg cells harvested from all other cultures, and furthermore inhibited acute graft versus host disease and especially skin transplant rejection. Thus, addition of TGF-β+RA seems to be superior over Rapa in stabilising the phenotype and functional capacity of aTreg cells.
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http://dx.doi.org/10.1002/eji.201243292DOI Listing
December 2013

Elevation of CD4+ differentiated memory T cells is associated with acute cellular and antibody-mediated rejection after liver transplantation.

Transplantation 2013 Jun;95(12):1512-20

Department of Visceral- and Transplant Surgery, Charite Universitätsmedizin, Augustenburger Platz 1, Berlin, Germany.

Background: It is now well known that the outcome after allogeneic transplantation, such as incidence of acute rejections, very much depends on the individual's immune reactivity status. There is also increasing evidence that the presence of preexisting memory T cells can affect antigraft immune responses.

Methods: In a prospective study, we monitored peripheral CD4 and CD8 central memory, effector memory, and terminal differentiated effector memory (TEMRA) T cells in 55 patients who underwent deceased liver transplantation and received conventional immunosuppressive treatment with or without basiliximab induction. The primary endpoint of the study was acute allograft rejection during a 1-year follow-up period.

Results: We observed significantly increased proportions of CD4 and CD8 TEMRA cells in patients before transplantation compared with healthy controls (P=0.006 and 0.009, respectively). This characteristic was independent of the underlying disease. In patients with no signs of acute rejection, we observed an immediate reduction of CD4 TEMRA cells. In contrast, patients who experienced acute cellular rejection, and especially antibody-mediated rejection, displayed persistent elevated TEMRA cells (P=0.017 and 0.027, respectively). Basiliximab induction therapy did not influence CD4 and CD8 TEMRA numbers.

Conclusions: Conventional immunosuppressive or basiliximab treatment cannot control the persistence of TEMRA T cells, which may contribute to acute cellular rejection and antibody-mediated rejection after liver transplantation. In the future, specific targeting of TEMRA cells in selected patients may prevent the occurrence of difficult to treat steroid-resistant rejections, thereby leading to improved patient outcome.
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http://dx.doi.org/10.1097/TP.0b013e318290de18DOI Listing
June 2013