Publications by authors named "Désirée Kunkel"

38 Publications

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

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 colonic mucosa-associated microbiome in SIV infection: shift towards Bacteroidetes coincides with mucosal CD4 T cell depletion and enterocyte damage.

Sci Rep 2020 07 2;10(1):10887. Epub 2020 Jul 2.

Institute of Medical Virology, Charité-Universitätsmedizin Berlin, Campus Mitte, 10117, Berlin, Germany.

The intesinal microbiome is considered important in human immunodeficiency virus (HIV) pathogenesis and therefore represents a potential therapeutic target to improve the patients' health status. Longitudinal alterations in the colonic mucosa-associated microbiome during simian immunodeficiency virus (SIV) infection were investigated using a 16S rRNA amplicon approach on the Illumina sequencing platform and bioinformatics analyses. Following SIV infection of six animals, no alterations in microbial composition were observed before the viral load peaked in the colon. At the time of acute mucosal SIV replication, the phylum Bacteroidetes including the Bacteroidia class as well as the phylum Firmicutes and its families Ruminococcaceae and Eubacteriaceae became more abundant. Enrichment of Bacteroidetes was maintained until the chronic phase of SIV infection. The shift towards Bacteroidetes in the mucosa-associated microbiome was associated with the extent of SIV infection-induced mucosal CD4 T cell depletion and correlated with increasing rates of enterocyte damage. These observations suggest that Bacteroidetes strains increase during virus-induced mucosal immune destruction. As Bacteroidetes belong to the lipopolysaccharide- and short chain fatty acids-producing bacteria, their rapid enrichment may contribute to inflammatory tissue damage and metabolic alterations in SIV/HIV infection. These aspects should be considered in future studies on therapeutic interventions.
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http://dx.doi.org/10.1038/s41598-020-67843-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7331662PMC
July 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

Leptin induces TNFα-dependent inflammation in acquired generalized lipodystrophy and combined Crohn's disease.

Nat Commun 2019 12 10;10(1):5629. Epub 2019 Dec 10.

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

Leptin has been shown to modulate intestinal inflammation in mice. However, clinical evidence regarding its immune-stimulatory potential in human Crohn's disease remains sparse. We here describe a patient with the unique combination of acquired generalized lipodystrophy and Crohn's disease (AGLCD) featuring a lack of adipose tissue, leptin deficiency and intestinal inflammation. Using mass and flow cytometry, immunohistochemistry and functional metabolic analyses, the AGLCD patient was compared to healthy individuals and Crohn's disease patients regarding immune cell composition, function and metabolism and the effects of recombinant N-methionylleptin (rLeptin) were evaluated. We provide evidence that rLeptin exerts diverse pro-inflammatory effects on immune cell differentiation and function, including the metabolic reprogramming of immune cells and the induction of TNFα, ultimately aggravating Crohn's disease in the AGLCD patient, which can be reversed by anti-TNFα therapy. Our results indicate that leptin is required for human immune homeostasis and contributes to autoimmunity in a TNFα-dependent manner.
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http://dx.doi.org/10.1038/s41467-019-13559-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6904732PMC
December 2019

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition).

Authors:
Andrea Cossarizza Hyun-Dong Chang Andreas Radbruch Andreas Acs Dieter Adam Sabine Adam-Klages William W Agace Nima Aghaeepour Mübeccel Akdis Matthieu Allez Larissa Nogueira Almeida Giorgia Alvisi Graham Anderson Immanuel Andrä Francesco Annunziato Achille Anselmo Petra Bacher Cosima T Baldari Sudipto Bari Vincenzo Barnaba Joana Barros-Martins Luca Battistini Wolfgang Bauer Sabine Baumgart Nicole Baumgarth Dirk Baumjohann Bianka Baying Mary Bebawy Burkhard Becher Wolfgang Beisker Vladimir Benes Rudi Beyaert Alfonso Blanco Dominic A Boardman Christian Bogdan Jessica G Borger Giovanna Borsellino Philip E Boulais Jolene A Bradford Dirk Brenner Ryan R Brinkman Anna E S Brooks Dirk H Busch Martin Büscher Timothy P Bushnell Federica Calzetti Garth Cameron Ilenia Cammarata Xuetao Cao Susanna L Cardell Stefano Casola Marco A Cassatella Andrea Cavani Antonio Celada Lucienne Chatenoud Pratip K Chattopadhyay Sue Chow Eleni Christakou Luka Čičin-Šain Mario Clerici Federico S Colombo Laura Cook Anne Cooke Andrea M Cooper Alexandra J Corbett Antonio Cosma Lorenzo Cosmi Pierre G Coulie Ana Cumano Ljiljana Cvetkovic Van Duc Dang Chantip Dang-Heine Martin S Davey Derek Davies Sara De Biasi Genny Del Zotto Gelo Victoriano Dela Cruz Michael Delacher Silvia Della Bella Paolo Dellabona Günnur Deniz Mark Dessing James P Di Santo Andreas Diefenbach Francesco Dieli Andreas Dolf Thomas Dörner Regine J Dress Diana Dudziak Michael Dustin Charles-Antoine Dutertre Friederike Ebner Sidonia B G Eckle Matthias Edinger Pascale Eede Götz R A Ehrhardt Marcus Eich Pablo Engel Britta Engelhardt Anna Erdei Charlotte Esser Bart Everts Maximilien Evrard Christine S Falk Todd A Fehniger Mar Felipo-Benavent Helen Ferry Markus Feuerer Andrew Filby Kata Filkor Simon Fillatreau Marie Follo Irmgard Förster John Foster Gemma A Foulds Britta Frehse Paul S Frenette Stefan Frischbutter Wolfgang Fritzsche David W Galbraith Anastasia Gangaev Natalio Garbi Brice Gaudilliere Ricardo T Gazzinelli Jens Geginat Wilhelm Gerner Nicholas A Gherardin Kamran Ghoreschi Lara Gibellini Florent Ginhoux Keisuke Goda Dale I Godfrey Christoph Goettlinger Jose M González-Navajas Carl S Goodyear Andrea Gori Jane L Grogan Daryl Grummitt Andreas Grützkau Claudia Haftmann Jonas Hahn Hamida Hammad Günter Hämmerling Leo Hansmann Goran Hansson Christopher M Harpur Susanne Hartmann Andrea Hauser Anja E Hauser David L Haviland David Hedley Daniela C Hernández Guadalupe Herrera Martin Herrmann Christoph Hess Thomas Höfer Petra Hoffmann Kristin Hogquist Tristan Holland Thomas Höllt Rikard Holmdahl Pleun Hombrink Jessica P Houston Bimba F Hoyer Bo Huang Fang-Ping Huang Johanna E Huber Jochen Huehn Michael Hundemer Christopher A Hunter William Y K Hwang Anna Iannone Florian Ingelfinger Sabine M Ivison Hans-Martin Jäck Peter K Jani Beatriz Jávega Stipan Jonjic Toralf Kaiser Tomas Kalina Thomas Kamradt Stefan H E Kaufmann Baerbel Keller Steven L C Ketelaars Ahad Khalilnezhad Srijit Khan Jan Kisielow Paul Klenerman Jasmin Knopf Hui-Fern Koay Katja Kobow Jay K Kolls Wan Ting Kong Manfred Kopf Thomas Korn Katharina Kriegsmann Hendy Kristyanto Thomas Kroneis Andreas Krueger Jenny Kühne Christian Kukat Désirée Kunkel Heike Kunze-Schumacher Tomohiro Kurosaki Christian Kurts Pia Kvistborg Immanuel Kwok Jonathan Landry Olivier Lantz Paola Lanuti Francesca LaRosa Agnès Lehuen Salomé LeibundGut-Landmann Michael D Leipold Leslie Y T Leung Megan K Levings Andreia C Lino Francesco Liotta Virginia Litwin Yanling Liu Hans-Gustaf Ljunggren Michael Lohoff Giovanna Lombardi Lilly Lopez Miguel López-Botet Amy E Lovett-Racke Erik Lubberts Herve Luche Burkhard Ludewig Enrico Lugli Sebastian Lunemann Holden T Maecker Laura Maggi Orla Maguire Florian Mair Kerstin H Mair Alberto Mantovani Rudolf A Manz Aaron J Marshall Alicia Martínez-Romero Glòria Martrus Ivana Marventano Wlodzimierz Maslinski Giuseppe Matarese Anna Vittoria Mattioli Christian Maueröder Alessio Mazzoni James McCluskey Mairi McGrath Helen M McGuire Iain B McInnes Henrik E Mei Fritz Melchers Susanne Melzer Dirk Mielenz Stephen D Miller Kingston H G Mills Hans Minderman Jenny Mjösberg Jonni Moore Barry Moran Lorenzo Moretta Tim R Mosmann Susann Müller Gabriele Multhoff Luis Enrique Muñoz Christian Münz Toshinori Nakayama Milena Nasi Katrin Neumann Lai Guan Ng Antonia Niedobitek Sussan Nourshargh Gabriel Núñez José-Enrique O'Connor Aaron Ochel Anna Oja Diana Ordonez Alberto Orfao Eva Orlowski-Oliver Wenjun Ouyang Annette Oxenius Raghavendra Palankar Isabel Panse Kovit Pattanapanyasat Malte Paulsen Dinko Pavlinic Livius Penter Pärt Peterson Christian Peth Jordi Petriz Federica Piancone Winfried F Pickl Silvia Piconese Marcello Pinti A Graham Pockley Malgorzata Justyna Podolska Zhiyong Poon Katharina Pracht Immo Prinz Carlo E M Pucillo Sally A Quataert Linda Quatrini Kylie M Quinn Helena Radbruch Tim R D J Radstake Susann Rahmig Hans-Peter Rahn Bartek Rajwa Gevitha Ravichandran Yotam Raz Jonathan A Rebhahn Diether Recktenwald Dorothea Reimer Caetano Reis e Sousa Ester B M Remmerswaal Lisa Richter Laura G Rico Andy Riddell Aja M Rieger J Paul Robinson Chiara Romagnani Anna Rubartelli Jürgen Ruland Armin Saalmüller Yvan Saeys Takashi Saito Shimon Sakaguchi Francisco Sala-de-Oyanguren Yvonne Samstag Sharon Sanderson Inga Sandrock Angela Santoni Ramon Bellmàs Sanz Marina Saresella Catherine Sautes-Fridman Birgit Sawitzki Linda Schadt Alexander Scheffold Hans U Scherer Matthias Schiemann Frank A Schildberg Esther Schimisky Andreas Schlitzer Josephine Schlosser Stephan Schmid Steffen Schmitt Kilian Schober Daniel Schraivogel Wolfgang Schuh Thomas Schüler Reiner Schulte Axel Ronald Schulz Sebastian R Schulz Cristiano Scottá Daniel Scott-Algara David P Sester T Vincent Shankey Bruno Silva-Santos Anna Katharina Simon Katarzyna M Sitnik Silvano Sozzani Daniel E Speiser Josef Spidlen Anders Stahlberg Alan M Stall Natalie Stanley Regina Stark Christina Stehle Tobit Steinmetz Hannes Stockinger Yousuke Takahama Kiyoshi Takeda Leonard Tan Attila Tárnok Gisa Tiegs Gergely Toldi Julia Tornack Elisabetta Traggiai Mohamed Trebak Timothy I M Tree Joe Trotter John Trowsdale Maria Tsoumakidou Henning Ulrich Sophia Urbanczyk Willem van de Veen Maries van den Broek Edwin van der Pol Sofie Van Gassen Gert Van Isterdael René A W van Lier Marc Veldhoen Salvador Vento-Asturias Paulo Vieira David Voehringer Hans-Dieter Volk Anouk von Borstel Konrad von Volkmann Ari Waisman Rachael V Walker Paul K Wallace Sa A Wang Xin M Wang Michael D Ward Kirsten A Ward-Hartstonge Klaus Warnatz Gary Warnes Sarah Warth Claudia Waskow James V Watson Carsten Watzl Leonie Wegener Thomas Weisenburger Annika Wiedemann Jürgen Wienands Anneke Wilharm Robert John Wilkinson Gerald Willimsky James B Wing Rieke Winkelmann Thomas H Winkler Oliver F Wirz Alicia Wong Peter Wurst Jennie H M Yang Juhao Yang Maria Yazdanbakhsh Liping Yu Alice Yue Hanlin Zhang Yi Zhao Susanne Maria Ziegler Christina Zielinski Jakob Zimmermann Arturo Zychlinsky

Eur J Immunol 2019 Oct;49(10):1457-1973

Max Planck Institute for Infection Biology, Berlin, Germany.

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.
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http://dx.doi.org/10.1002/eji.201970107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7350392PMC
October 2019

Cell type-dependent differential activation of ERK by oncogenic KRAS in colon cancer and intestinal epithelium.

Nat Commun 2019 07 2;10(1):2919. Epub 2019 Jul 2.

Institute of Pathology, Charité Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany.

Oncogenic mutations in KRAS or BRAF are frequent in colorectal cancer and activate the ERK kinase. Here, we find graded ERK phosphorylation correlating with cell differentiation in patient-derived colorectal cancer organoids with and without KRAS mutations. Using reporters, single cell transcriptomics and mass cytometry, we observe cell type-specific phosphorylation of ERK in response to transgenic KRAS in mouse intestinal organoids, while transgenic BRAF activates ERK in all cells. Quantitative network modelling from perturbation data reveals that activation of ERK is shaped by cell type-specific MEK to ERK feed forward and negative feedback signalling. We identify dual-specificity phosphatases as candidate modulators of ERK in the intestine. Furthermore, we find that oncogenic KRAS, together with β-Catenin, favours expansion of crypt cells with high ERK activity. Our experiments highlight key differences between oncogenic BRAF and KRAS in colorectal cancer and find unexpected heterogeneity in a signalling pathway with fundamental relevance for cancer therapy.
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http://dx.doi.org/10.1038/s41467-019-10954-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6606648PMC
July 2019

Setting Up Mass Cytometry in a Shared Resource Lab Environment.

Methods Mol Biol 2019 ;1989:3-11

BCRT Flow Cytometry Lab, Berlin-Brandenburg Center for Regenerative Therapies, Charité - Universitätsmedizin Berlin, Berlin, Germany.

Core facilities or shared resource laboratories (SRL) ensure fast and direct access to high-end instrumentation that is often expensive and requires technical expertise to be operated and maintained. SRL have the responsibility and capability to ensure the possibility of generating reproducible quality data by implementing best practices, providing expert knowledge, and keeping the necessary instrumentation in good shape. Here we review critical steps involved in the integration of mass cytometry in a shared resource lab environment. Lab requirements and considerations for best practices are discussed, and critical aspects of mass cytometry project discussions as well as strategies for the management and documentation of samples are presented.
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http://dx.doi.org/10.1007/978-1-4939-9454-0_1DOI Listing
March 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

The IL-1 Pathway Is Hyperactive in Hidradenitis Suppurativa and Contributes to Skin Infiltration and Destruction.

J Invest Dermatol 2019 06 5;139(6):1294-1305. Epub 2018 Dec 5.

Interdisciplinary Group Molecular Immunopathology, Dermatology/Medical Immunology, Charité-Universitätsmedizin Berlin, Berlin, Germany; Psoriasis Research and Treatment Center, Charité-Universitätsmedizin Berlin, Berlin, Germany. Electronic address:

Hidradenitis suppurativa (HS) (also designated acne inversa) is a chronic inflammatory disease characterized by painful purulent skin lesions and progressive destruction of skin architecture. Despite the high burden for the patients, pathogenetic pathways underlying HS alterations remain obscure. When we examined the HS cytokine pattern, IL-1β turned out to be a highly prominent cytokine, overexpressed even compared with psoriatic lesions. Analyses of IL-1β-induced transcriptome in various cell types showed overlapping profiles, with upregulations of molecules causing immune cell infiltration and extracellular matrix degradation, and of specific cytokines including IL-6, IL-32, and IL-36. Matching cellular IL-1 receptor levels, dermal fibroblasts showed both the strongest and broadest IL-1β response, which was not clearly shared or strengthened by other cytokines. The IL-1β signature was specifically present in HS lesions and could be reversed by application of IL-1 receptor antagonist. Search for blood parameters associated with IL-1β pathway activity in HS identified serum amyloid A, which was synergistically induced by IL-1β and IL-6 in hepatocytes. Consequently, strongly elevated blood serum amyloid A levels in HS correlated positively with the extent of inflammatory skin alterations. In summary, the IL-1β pathway represents a pathogenetic cascade, whose activity may be therapeutically targeted and monitored by blood SAA levels.
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http://dx.doi.org/10.1016/j.jid.2018.11.018DOI Listing
June 2019

Cell-Associated Simian Immunodeficiency Virus Accelerates Initial Virus Spread and CD4+ T-Cell Depletion in the Intestinal Mucosa.

J Infect Dis 2018 04;217(9):1421-1425

Department of Gastroenterology, Infectious Diseases, and Rheumatology.

Cell-free and cell-associated human immunodeficiency virus (HIV) may differently affect the immune system and the efficacy of prevention strategies. Here we examined mucosal events in simian immunodeficiency virus (SIV) infection, using infected cells together with cell-free virus and cell-free virus alone. Intravenously inoculated SIV-infected cells disseminated virus to the intestine within 16 hours. Infection with both virus forms accelerated viral dissemination in the intestinal mucosa and the loss of mucosal CD4+ T cells as compared to infection with cell-free virus only. As all natural sources of HIV infection contain both virus forms, future prevention studies should focus on efficacy against both cell-free and cell-associated virus.
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http://dx.doi.org/10.1093/infdis/jiy055DOI Listing
April 2018

Guidelines for the use of flow cytometry and cell sorting in immunological studies.

Authors:
Andrea Cossarizza Hyun-Dong Chang Andreas Radbruch Mübeccel Akdis Immanuel Andrä Francesco Annunziato Petra Bacher Vincenzo Barnaba Luca Battistini Wolfgang M Bauer Sabine Baumgart Burkhard Becher Wolfgang Beisker Claudia Berek Alfonso Blanco Giovanna Borsellino Philip E Boulais Ryan R Brinkman Martin Büscher Dirk H Busch Timothy P Bushnell Xuetao Cao Andrea Cavani Pratip K Chattopadhyay Qingyu Cheng Sue Chow Mario Clerici Anne Cooke Antonio Cosma Lorenzo Cosmi Ana Cumano Van Duc Dang Derek Davies Sara De Biasi Genny Del Zotto Silvia Della Bella Paolo Dellabona Günnur Deniz Mark Dessing Andreas Diefenbach James Di Santo Francesco Dieli Andreas Dolf Vera S Donnenberg Thomas Dörner Götz R A Ehrhardt Elmar Endl Pablo Engel Britta Engelhardt Charlotte Esser Bart Everts Anita Dreher Christine S Falk Todd A Fehniger Andrew Filby Simon Fillatreau Marie Follo Irmgard Förster John Foster Gemma A Foulds Paul S Frenette David Galbraith Natalio Garbi Maria Dolores García-Godoy Jens Geginat Kamran Ghoreschi Lara Gibellini Christoph Goettlinger Carl S Goodyear Andrea Gori Jane Grogan Mor Gross Andreas Grützkau Daryl Grummitt Jonas Hahn Quirin Hammer Anja E Hauser David L Haviland David Hedley Guadalupe Herrera Martin Herrmann Falk Hiepe Tristan Holland Pleun Hombrink Jessica P Houston Bimba F Hoyer Bo Huang Christopher A Hunter Anna Iannone Hans-Martin Jäck Beatriz Jávega Stipan Jonjic Kerstin Juelke Steffen Jung Toralf Kaiser Tomas Kalina Baerbel Keller Srijit Khan Deborah Kienhöfer Thomas Kroneis Désirée Kunkel Christian Kurts Pia Kvistborg Joanne Lannigan Olivier Lantz Anis Larbi Salome LeibundGut-Landmann Michael D Leipold Megan K Levings Virginia Litwin Yanling Liu Michael Lohoff Giovanna Lombardi Lilly Lopez Amy Lovett-Racke Erik Lubberts Burkhard Ludewig Enrico Lugli Holden T Maecker Glòria Martrus Giuseppe Matarese Christian Maueröder Mairi McGrath Iain McInnes Henrik E Mei Fritz Melchers Susanne Melzer Dirk Mielenz Kingston Mills David Mirrer Jenny Mjösberg Jonni Moore Barry Moran Alessandro Moretta Lorenzo Moretta Tim R Mosmann Susann Müller Werner Müller Christian Münz Gabriele Multhoff Luis Enrique Munoz Kenneth M Murphy Toshinori Nakayama Milena Nasi Christine Neudörfl John Nolan Sussan Nourshargh José-Enrique O'Connor Wenjun Ouyang Annette Oxenius Raghav Palankar Isabel Panse Pärt Peterson Christian Peth Jordi Petriz Daisy Philips Winfried Pickl Silvia Piconese Marcello Pinti A Graham Pockley Malgorzata Justyna Podolska Carlo Pucillo Sally A Quataert Timothy R D J Radstake Bartek Rajwa Jonathan A Rebhahn Diether Recktenwald Ester B M Remmerswaal Katy Rezvani Laura G Rico J Paul Robinson Chiara Romagnani Anna Rubartelli Beate Ruckert Jürgen Ruland Shimon Sakaguchi Francisco Sala-de-Oyanguren Yvonne Samstag Sharon Sanderson Birgit Sawitzki Alexander Scheffold Matthias Schiemann Frank Schildberg Esther Schimisky Stephan A Schmid Steffen Schmitt Kilian Schober Thomas Schüler Axel Ronald Schulz Ton Schumacher Cristiano Scotta T Vincent Shankey Anat Shemer Anna-Katharina Simon Josef Spidlen Alan M Stall Regina Stark Christina Stehle Merle Stein Tobit Steinmetz Hannes Stockinger Yousuke Takahama Attila Tarnok ZhiGang Tian Gergely Toldi Julia Tornack Elisabetta Traggiai Joe Trotter Henning Ulrich Marlous van der Braber René A W van Lier Marc Veldhoen Salvador Vento-Asturias Paulo Vieira David Voehringer Hans-Dieter Volk Konrad von Volkmann Ari Waisman Rachael Walker Michael D Ward Klaus Warnatz Sarah Warth James V Watson Carsten Watzl Leonie Wegener Annika Wiedemann Jürgen Wienands Gerald Willimsky James Wing Peter Wurst Liping Yu Alice Yue Qianjun Zhang Yi Zhao Susanne Ziegler Jakob Zimmermann

Eur J Immunol 2017 10;47(10):1584-1797

Maurice Müller Laboratories (DKF), Universitätsklinik für Viszerale Chirurgie und Medizin Inselspital, University of Bern, Murtenstrasse, Bern.

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http://dx.doi.org/10.1002/eji.201646632DOI Listing
October 2017

International Society for Advancement of Cytometry (ISAC) flow cytometry shared resource laboratory (SRL) best practices.

Cytometry A 2016 11 3;89(11):1017-1030. Epub 2016 Nov 3.

Flow Cytometry Core Facility, Babraham Institute, Cambridge, United Kingdom.

The purpose of this document is to define minimal standards for a flow cytometry shared resource laboratory (SRL) and provide guidance for best practices in several important areas. This effort is driven by the desire of International Society for the Advancement of Cytometry (ISAC) members in SRLs to define and maintain standards of excellence in flow cytometry, and act as a repository for key elements of this information (e.g. example SOPs/training material, etc.). These best practices are not intended to define specifically how to implement these recommendations, but rather to establish minimal goals for an SRL to address in order to achieve excellence. It is hoped that once these best practices are established and implemented they will serve as a template from which similar practices can be defined for other types of SRLs. Identification of the need for best practices first occurred through discussions at the CYTO 2013 SRL Forum, with the most important areas for which best practices should be defined identified through several surveys and SRL track workshops as part of CYTO 2014. © 2016 International Society for Advancement of Cytometry.
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http://dx.doi.org/10.1002/cyto.a.23016DOI 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

Modulation of type I interferon-associated viral sensing during acute simian immunodeficiency virus infection in African green monkeys.

J Virol 2015 Jan 29;89(1):751-62. Epub 2014 Oct 29.

Institut Pasteur, Unité de Régulation des Infections Rétrovirales, Paris, France

Unlabelled: Natural hosts of simian immunodeficiency virus (SIV), such as African green monkeys (AGMs), do not progress to AIDS when infected with SIV. This is associated with an absence of a chronic type I interferon (IFN-I) signature. It is unclear how the IFN-I response is downmodulated in AGMs. We longitudinally assessed the capacity of AGM blood cells to produce IFN-I in response to SIV and herpes simplex virus (HSV) infection. Phenotypes and functions of plasmacytoid dendritic cells (pDCs) and other mononuclear blood cells were assessed by flow cytometry, and expression of viral sensors was measured by reverse transcription-PCR. pDCs displayed low BDCA-2, CD40, and HLA-DR expression levels during AGM acute SIV (SIVagm) infection. BDCA-2 was required for sensing of SIV, but not of HSV, by pDCs. In acute infection, AGM peripheral blood mononuclear cells (PBMCs) produced less IFN-I upon SIV stimulation. In the chronic phase, the production was normal, confirming that the lack of chronic inflammation is not due to a sensing defect of pDCs. In contrast to stimulation by SIV, more IFN-I was produced upon HSV stimulation of PBMCs isolated during acute infection, while the frequency of AGM pDCs producing IFN-I upon in vitro stimulation with HSV was diminished. Indeed, other cells started producing IFN-I. This increased viral sensing by non-pDCs was associated with an upregulation of Toll-like receptor 3 and IFN-γ-inducible protein 16 caused by IFN-I in acute SIVagm infection. Our results suggest that, as in pathogenic SIVmac infection, SIVagm infection mobilizes bone marrow precursor pDCs. Moreover, we show that SIV infection modifies the capacity of viral sensing in cells other than pDCs, which could drive IFN-I production in specific settings.

Importance: The effects of HIV/SIV infections on the capacity of plasmacytoid dendritic cells (pDCs) to produce IFN-I in vivo are still incompletely defined. As IFN-I can restrict viral replication, contribute to inflammation, and influence immune responses, alteration of this capacity could impact the viral reservoir size. We observed that even in nonpathogenic SIV infection, the frequency of pDCs capable of efficiently sensing SIV and producing IFN-I was reduced during acute infection. We discovered that, concomitantly, cells other than pDCs had increased abilities for viral sensing. Our results suggest that pDC-produced IFN-I upregulates viral sensors in bystander cells, the latter gaining the capacity to produce IFN-I. These results indicate that in certain settings, cells other than pDCs can drive IFN-I-associated inflammation in SIV infection. This has important implications for the understanding of persistent inflammation and the establishment of viral reservoirs.
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http://dx.doi.org/10.1128/JVI.02430-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4301160PMC
January 2015

Modulation of NADPH oxidase activity by known uraemic retention solutes.

Eur J Clin Invest 2014 Aug;44(8):802-11

Charité-Universitätsmedizin Berlin (CBF), Medizinische Klinik IV, Berlin, Germany.

Background: Uraemia and cardiovascular disease appear to be associated with an increased oxidative burden. One of the key players in the genesis of reactive oxygen species (ROS) is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Based on initial experiments demonstrating a decreased inhibitory effect on NADPH oxidase activity in the presence of plasma from patients with CKD-5D after dialysis compared with before dialysis, we investigated the effect of 48 known and commercially available uraemic retention solutes on the enzymatic activity of NADPH oxidase.

Methods: Mononuclear leucocytes isolated from buffy coats of healthy volunteers were isolated, lysed and incubated with NADH in the presence of plasma from healthy controls and patients with CKD-5D. Furthermore, the leucocytes were lysed and incubated in the presence of uraemic retention solute of interest and diphenyleneiodonium chloride (DPI), an inhibitor of NADPH oxidase. The effect on enzymatic activity of NADPH oxidase was quantified within an incubation time of 120 min.

Results: Thirty-nine of the 48 uraemic retention solutes tested had a significant decreasing effect on NADPH oxidase activity. Oxalate has been characterized as the strongest inhibitor of NADPH oxidase (90% of DPI inhibition). Surprisingly, none of the uraemic retention solutes we investigated was found to increase NADPH oxidase activity. Furthermore, plasma from patients with CKD-5D before dialysis caused significantly higher inhibitory effect on NADPH oxidase activity compared with plasma from healthy subjects. However, this effect was significantly decreased in plasma from patients with CKD-5D after dialysis.

Conclusions: The results of this study show that uraemic retention solutes modulated the activity of the NADPH oxidase. The results of this study might be the basis for the development of inhibitors applicable as drug in the situation of increased oxidative stress.
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http://dx.doi.org/10.1111/eci.12297DOI Listing
August 2014

Innate immune responses and rapid control of inflammation in African green monkeys treated or not with interferon-alpha during primary SIVagm infection.

PLoS Pathog 2014 Jul 3;10(7):e1004241. Epub 2014 Jul 3.

Institut Pasteur, Regulation of Retroviral Infection Unit, Paris, France.

Chronic immune activation (IA) is considered as the driving force of CD4(+) T cell depletion and AIDS. Fundamental clues in the mechanisms that regulate IA could lie in natural hosts of SIV, such as African green monkeys (AGMs). Here we investigated the role of innate immune cells and IFN-α in the control of IA in AGMs. AGMs displayed significant NK cell activation upon SIVagm infection, which was correlated with the levels of IFN-α. Moreover, we detected cytotoxic NK cells in lymph nodes during the early acute phase of SIVagm infection. Both plasmacytoid and myeloid dendritic cell (pDC and mDC) homing receptors were increased, but the maturation of mDCs, in particular of CD16+ mDCs, was more important than that of pDCs. Monitoring of 15 cytokines showed that those, which are known to be increased early in HIV-1/SIVmac pathogenic infections, such as IL-15, IFN-α, MCP-1 and CXCL10/IP-10, were significantly increased in AGMs as well. In contrast, cytokines generally induced in the later stage of acute pathogenic infection, such as IL-6, IL-18 and TNF-α, were less or not increased, suggesting an early control of IA. We then treated AGMs daily with high doses of IFN-α from day 9 to 24 post-infection. No impact was observed on the activation or maturation profiles of mDCs, pDCs and NK cells. There was also no major difference in T cell activation or interferon-stimulated gene (ISG) expression profiles and no sign of disease progression. Thus, even after administration of high levels of IFN-α during acute infection, AGMs were still able to control IA, showing that IA control is independent of IFN-α levels. This suggests that the sustained ISG expression and IA in HIV/SIVmac infections involves non-IFN-α products.
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http://dx.doi.org/10.1371/journal.ppat.1004241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4081777PMC
July 2014

Effect of age on the CD4⁺ T-cell impairment in HIV-infected persons without and with cART.

J Acquir Immune Defic Syndr 2014 May;66(1):7-15

*Department of Gastroenterology, Infectious Diseases, and Rheumatology, Medical Clinic I, Campus Benjamin Franklin, Charité-University Medicine Berlin, Berlin, Germany; and †Ärzteforum Seestraβe, Berlin, Germany (Heiko Karcher is now with Ärzteforum Praxis City Ost, Berlin, Germany; Desiree Kunkel is now with Berlin-Brandenburg Center for Regenerative Therapies, Campus Virchow Klinikum, Charité-University Medicine Berlin, Berlin, Germany).

Background: Knowledge about HIV infection in older persons is becoming increasingly important. CD4⁺ T cells are essential for protective immunity, but little is known about the effect of age on the CD4⁺ T-cell impairment in HIV infection.

Methods: Treatment-naive patients aged older than 50 or younger than 40 years were studied for absolute and relative frequencies of CD31⁺ naive and CD31⁻ naive CD4⁺ T cells, central memory, effector memory, and terminally differentiated CD4⁺ T cells, and compared with age-matched controls. In addition, cellular proliferation and cytokine secretion properties were determined. CD4⁺ T-cell reconstitution was analyzed in older and younger patients with <350 or ≥ 350 CD4⁺ T cells per microliter at initiation of combination antiretroviral therapy (cART).

Results: CD4⁺ T cells of older but not younger HIV-infected patients showed age-inappropriate low levels of CD31⁻ naive cells, increased levels of effector memory cells, and enhanced interferonγ and interleukin-17 secretion. Impaired CD4⁺ T-cell composition persisted in patients who initiated cART at <350 CD4⁺ T cells per microliter. In patients with CD4⁺ T cells ≥ 350 per microliter, alterations were less pronounced and were reversible with cART. Compared with age-matched controls, total CD4⁺ T-cell counts did not differ between treated younger and older HIV-infected patients.

Conclusions: These data demonstrate that aging enhances the CD4⁺ T-cell impairment in HIV-infected persons mainly by a loss of CD31⁻ naive cells, accumulation of effector memory cells, and increased pro-inflammatory effector functions. Age-related changes in CD4⁺ T-cell composition can be prevented by an early initiation of cART.
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http://dx.doi.org/10.1097/QAI.0000000000000097DOI Listing
May 2014

Epithelial-to-Mesenchymal Transition Enhances the Cardioprotective Capacity of Human Amniotic Epithelial Cells.

Cell Transplant 2015 20;24(6):985-1002. Epub 2013 Nov 20.

Berlin Brandenburg Center for Regenerative Therapies, Berlin, Germany.

The amniotic epithelium consists of cells exhibiting mature epithelial cell characteristics, but also varying degrees of stemness. We tested the hypothesis that induction of epithelial-to-mesenchymal transition (EMT) in amniotic epithelial cells (AECs) derived from human placenta enhances their capacity to support the ischemic myocardium. In response to incubation with transforming growth factor-β1 (TGF-β1) protein, AECs lost their cobblestone morphology and acquired a fibroblastoid shape, associated with downregulation of E-cadherin, upregulation of N-cadherin, Akt phosphorylation, and intracellular periostin translocation. EMT-AECs displayed greatly enhanced mobility and secreted gelatinase activity compared with naive AECs. The surface presentation of CD105 and CD73 decreased, and RNA microarray analysis mirrored the loss of epithelial characteristics and transcriptional profile. Unmodified AECs and EMT-AECs were then injected intramyocardially in fully immunocompetent mice after permanent LAD ligation, and heart function was followed by MRI as well as 2D speckle tracking echocardiography after 4 weeks. EMT-AEC-treated infarct hearts displayed better global systolic function and improved longitudinal strain rate in the area of interest. Although no signals of human cells were detectable by histology, infarct size was smaller in EMT-AEC-treated hearts, associated with fewer TUNEL-positive cells and upregulation of periostin, while blood vessel density was increased in both ACE- and EMT-AEC-treated hearts. We conclude that EMT enhances the cardioprotective effects of human AECs.
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http://dx.doi.org/10.3727/096368913X675151DOI Listing
March 2016

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

Rabbit antithymocyte globulin (thymoglobulin) impairs the thymic output of both conventional and regulatory CD4+ T cells after allogeneic hematopoietic stem cell transplantation in adult patients.

Haematologica 2013 Jan 16;98(1):23-30. Epub 2012 Jul 16.

Department of Hematology, Oncology and Tumor Immunology, Charité, Berlin, Germany.

Rabbit antithymocyte globulin-Genzyme™ is used to prevent graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. Common disadvantages of treatment are infectious complications. The effects of rabbit antithymocyte globulin-Genzyme™ on thymic function have not been well-studied. Multicolor flow cytometry was used to analyze the kinetics of conventional and regulatory T cells in adult patients treated (n=12) or not treated (n=8) with rabbit antithymocyte globulin-Genzyme™ during the first 6 months after allogeneic hematopoietic stem cell transplantation. Patients treated with rabbit antithymocyte globulin-Genzyme™ had almost undetectable levels of recent thymic emigrants (CD45RA(+)CD31(+)) of both conventional and regulatory CD4T cells throughout the 6 months after allogeneic hematopoietic stem cell transplantation whereas CD4(+)CD45RA-memory T cells were less affected, but their levels were also significantly lower than in patients not treated with rabbit antithymocyte globulin-Genzyme™. In vitro, rabbit antithymocyte globulin-Genzyme™ induced apoptosis and cytolysis of human thymocytes, and its cytotoxic effects were greater than those of rabbit antithymocyte globulin-Fresenius™. Rabbit antithymocyte globulin-Genzyme™ in combination with a conditioning regimen strongly impairs thymic recovery of both conventional and regulatory CD4(+) T cells. The sustained depletion of conventional and regulatory CD4(+)T cells carries a high risk of both infections and graft-versus-host disease. Our data indicate that patients treated with rabbit antithymocyte globulin-Genzyme™ could benefit from thymus-protective therapies and that trials comparing this product with other rabbit antithymocyte globulin preparations or lymphocyte-depleting compounds would be informative.
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http://dx.doi.org/10.3324/haematol.2012.067611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3533656PMC
January 2013

Gut mucosal FOXP3+ regulatory CD4+ T cells and Nonregulatory CD4+ T cells are differentially affected by simian immunodeficiency virus infection in rhesus macaques.

J Virol 2010 Apr 13;84(7):3259-69. Epub 2010 Jan 13.

Medical Clinic I-Gastroenterology, Infectious Diseases and Rheumatology, Charité-Campus Benjamin Franklin, Hindenburgdamm 30, 12203 Berlin, Germany.

The gastrointestinal tract represents a major site for human and simian immunodeficiency virus (HIV and SIV) replication and CD4(+) T-cell depletion. Despite severe depletion of mucosal CD4(+) T cells, FOXP3(+) regulatory CD4(+) T cells (T(reg)) are highly increased in the gut mucosa of chronically HIV-infected individuals and may contribute to HIV pathogenesis, either by their immunosuppressive function or as a significant target cell population for virus production. Little is known about the susceptibility of mucosal T(reg) to viral infection and the longitudinal effect of HIV/SIV infection on T(reg) dynamics. In this study, we determined the level of SIV infection in T(reg) and nonregulatory CD4(+) T cells (non-T(reg)) isolated from the colon of SIV-infected rhesus macaques. The dynamics of mucosal T(reg) and alterations in the mucosal CD4(+) T-cell pool were examined longitudinally. Our findings indicate that mucosal T(reg) were less susceptible to productive SIV infection than non-T(reg) and thus were selectively spared from SIV-mediated cell death. In addition to improved survival, local expansion of T(reg) by SIV-induced proliferation of the mucosal CD4(+) T-cell pool facilitated the accumulation of mucosal T(reg) during the course of infection. High frequency of mucosal T(reg) in chronic SIV infection was strongly related to a reduction of perforin-expressing cells. In conclusion, this study suggests that mucosal T(reg) are less affected by productive SIV infection than non-T(reg) and therefore spared from depletion. Although SIV production is limited in mucosal T(reg), T(reg) accumulation may indirectly contribute to viral persistence by suppressing antiviral immune responses.
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http://dx.doi.org/10.1128/JVI.01715-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2838127PMC
April 2010

Nonpathogenic SIV infection of African green monkeys induces a strong but rapidly controlled type I IFN response.

J Clin Invest 2009 Dec;119(12):3544-55

Institut Pasteur, Unité de Régulation des Infections Rétrovirales, Paris, France.

African green monkeys (AGMs) infected with the AGM type of SIV (SIVagm) do not develop chronic immune activation and AIDS, despite viral loads similar to those detected in humans infected with HIV-1 and rhesus macaques (RMs) infected with the RM type of SIV (SIVmac). Because chronic immune activation drives progressive CD4+ T cell depletion and immune cell dysfunctions, factors that characterize disease progression, we sought to understand the molecular basis of this AGM phenotype. To this end, we longitudinally assessed the gene expression profiles of blood- and lymph node-derived CD4+ cells from AGMs and RMs in response to SIVagm and SIVmac infection, respectively, using a genomic microarray platform. The molecular signature of acute infection was characterized, in both species, by strong upregulation of type I IFN-stimulated genes (ISGs). ISG expression returned to basal levels after postinfection day 28 in AGMs but was sustained in RMs, especially in the lymph node-derived cells. We also found that SIVagm induced IFN-alpha production by AGM cells in vitro and that low IFN-alpha levels were sufficient to induce strong ISG responses. In conclusion, SIV infection triggered a rapid and strong IFN-alpha response in vivo in both AGMs and RMs, with this response being efficiently controlled only in AGMs, possibly as a result of active regulatory mechanisms.
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http://dx.doi.org/10.1172/JCI40093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2786805PMC
December 2009

Impaired immune functions of monocytes and macrophages in Whipple's disease.

Gastroenterology 2010 Jan 5;138(1):210-20. Epub 2009 Aug 5.

Medizinische Klinik I, Charité-Universitätsmedizin Berlin, CBF, Berlin, Germany.

Background & Aims: Whipple's disease is a chronic multisystemic infection caused by Tropheryma whipplei. Host factors likely predispose for the establishment of an infection, and macrophages seem to be involved in the pathogenesis of Whipple's disease. However, macrophage activation in Whipple's disease has not been studied systematically so far.

Methods: Samples from 145 Whipple's disease patients and 166 control subjects were investigated. We characterized duodenal macrophages and lymphocytes immunohistochemically and peripheral monocytes by flow cytometry and quantified mucosal and systemic cytokines and chemokines indicative for macrophage activation. In addition, we determined duodenal nitrite production and oxidative burst induced by T whipplei and by other bacteria.

Results: Reduced numbers of duodenal lymphocytes, increased numbers of CD163(+) and stabilin-1(+), reduced numbers of inducible nitric synthase+ duodenal macrophages, and increased percentages of CD163(+) peripheral monocytes indicated a lack of inflammation and a M2/alternatively activated macrophage phenotype in Whipple's disease. Incubation with T whipplei in vitro enhanced the expression of CD163 on monocytes from Whipple's disease patients but not from control subjects. Chemokines and cytokines associated with M2/alternative macrophage activation were elevated in the duodenum and the peripheral blood from Whipple's disease patients. Functionally, Whipple's disease patients showed a reduced duodenal nitrite production and reduced oxidative burst upon incubation with T whipplei compared with healthy subjects.

Conclusions: The lack of excessive local inflammation and alternative activation of macrophages, triggered in part by the agent T whipplei itself, may explain the hallmark of Whipple's disease: invasion of the intestinal mucosa with macrophages incompetent to degrade T whipplei.
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http://dx.doi.org/10.1053/j.gastro.2009.07.066DOI Listing
January 2010

Mutations in PYCR1 cause cutis laxa with progeroid features.

Nat Genet 2009 Sep 2;41(9):1016-21. Epub 2009 Aug 2.

Institute of Medical Biology, A*STAR, Singapore, Singapore.

Autosomal recessive cutis laxa (ARCL) describes a group of syndromal disorders that are often associated with a progeroid appearance, lax and wrinkled skin, osteopenia and mental retardation. Homozygosity mapping in several kindreds with ARCL identified a candidate region on chromosome 17q25. By high-throughput sequencing of the entire candidate region, we detected disease-causing mutations in the gene PYCR1. We found that the gene product, an enzyme involved in proline metabolism, localizes to mitochondria. Altered mitochondrial morphology, membrane potential and increased apoptosis rate upon oxidative stress were evident in fibroblasts from affected individuals. Knockdown of the orthologous genes in Xenopus and zebrafish led to epidermal hypoplasia and blistering that was accompanied by a massive increase of apoptosis. Our findings link mutations in PYCR1 to altered mitochondrial function and progeroid changes in connective tissues.
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http://dx.doi.org/10.1038/ng.413DOI Listing
September 2009

Migration patterns of nonspecifically activated versus nonactivated nonhuman primate T lymphocytes: preferential homing of activated autologous CD8+ T cells in the rectal mucosa.

J Immunother 2008 May;31(4):334-44

Medical Clinic I, Gastroenterology, Infectious Diseases and Rheumatology, Charité-Campus Benjamin Franklin, Berlin, Germany.

Adoptive cell transfer may be a successful strategy in anticancer therapy and its therapeutic efficiency depends on the access of transferred cells to the tumor site and their persistence in vivo. Nevertheless, the migration properties of autologous in vitro-activated T cells in primates are largely unknown. Here, we established the long-term tracking of T-cell migration into various compartments of rhesus macaques as a preclinical model for the evaluation of T-cell-based immunotherapy. Peripheral blood mononuclear cells from 3 to 4 rhesus macaques were activated with anti-CD3/anti-CD28 or not, labeled with carboxyfluorescein diacetat succinimidyl ester, and reinjected intravenously into the donor animals. Blood samples, lymph node biopsies, and mucosal biopsies (duodenum, rectum) were collected at various time points and analyzed by flow cytometry for the presence of the reinjected T cells. We demonstrate that nonspecific in vitro activation changes the in vivo migratory behavior of T cells and provokes a preferential migration of CD8 T cells to the rectum. Nonspecifically activated transferred CD4 T cells were found in much lower frequencies at this site and also in other compartments. Thus, our results indicate an imbalanced distribution of autologous CD8 and CD4 T cells in various compartments that is more apparent when T cells are activated before the transfer. The migratory behavior of in vitro-expanded, autologously transferred T cells can, therefore, influence the clinical outcome of adoptive cell transfer.
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http://dx.doi.org/10.1097/CJI.0b013e3181635e7fDOI Listing
May 2008

Plasmacytoid dendritic cell dynamics and alpha interferon production during Simian immunodeficiency virus infection with a nonpathogenic outcome.

J Virol 2008 Jun 2;82(11):5145-52. Epub 2008 Apr 2.

Institut Pasteur, Dakar, Sénégal.

We addressed the role of plasmacytoid dendritic cells (PDC) in protection against AIDS in nonpathogenic simian immunodeficiency virus (SIVagm) infection in African green monkeys (AGMs). PDC were monitored in blood and lymph nodes (LNs) starting from day 1 postinfection. We observed significant declines in blood during acute infection. However, PDC then returned to normal levels, and chronically infected AGMs showed no decrease of PDC in blood. There was a significant increase of PDC in LNs during acute infection. Blood PDC displayed only weak alpha interferon (IFN-alpha) responses to TLR9 agonist stimulation before infection. However, during acute infection, both blood and LN PDC showed a transiently increased propensity for IFN-alpha production. Bioactive IFN-alpha was detected in plasma concomitant with the peak of viremia, though levels were only low to moderate in some animals. Plasma interleukin 6 (IL-6) and IL-12 were not increased. In conclusion, PDC were recruited to the LNs and displayed increased IFN-alpha production during acute infection. However, increases in IFN-alpha were transient. Together with the lack of inflammatory cytokine responses, these events might play an important role in the low level of T-cell activation which is associated with protection against AIDS in nonpathogenic SIVagm infection.
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http://dx.doi.org/10.1128/JVI.02433-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2395206PMC
June 2008