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Article: Auf's Putzen verzichten : Modifizierte Sanger-Sequenzierung

Witter, Klaus

2011 Volume 18, Issue 10, Page(s) 88–89

Keywords	Sanger-Sequenzierung ; DNA Analyse ; Rationalisierung ; Arbeitsablauf
Language	German
Document type	Article
ZDB-ID	1237282-1
ISSN	1612-8354
ISSN	1612-8354
Database	bibnet.org

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Article ; Online: Immunoinformatic Analysis Reveals Antigenic Heterogeneity of Epstein-Barr Virus Is Immune-Driven.

Cirac, Ana / Poirey, Remy / Dieckmeyer, Michael / Witter, Klaus / Delecluse, Henri-Jacques / Behrends, Uta / Mautner, Josef

Frontiers in immunology

2021 Volume 12, Page(s) 796379

Abstract: Whole genome sequencing of Epstein-Barr virus (EBV) isolates from around the world has uncovered pervasive strain heterogeneity, but the forces driving strain diversification and the impact on immune recognition remained largely unknown. Using a data ... ...

Abstract	Whole genome sequencing of Epstein-Barr virus (EBV) isolates from around the world has uncovered pervasive strain heterogeneity, but the forces driving strain diversification and the impact on immune recognition remained largely unknown. Using a data mining approach, we analyzed more than 300 T-cell epitopes in 168 published EBV strains. Polymorphisms were detected in approximately 65% of all CD8+ and 80% of all CD4+ T-cell epitopes and these numbers further increased when epitope flanking regions were included. Polymorphisms in CD8+ T-cell epitopes often involved MHC anchor residues and resulted in changes of the amino acid subgroup, suggesting that only a limited number of conserved T-cell epitopes may represent generic target antigens against different viral strains. Although considered the prototypic EBV strain, the rather low degree of overlap with most other viral strains implied that B95.8 may not represent the ideal reference strain for T-cell epitopes. Instead, a combinatorial library of consensus epitopes may provide better targets for diagnostic and therapeutic purposes when the infecting strain is unknown. Polymorphisms were significantly enriched in epitope versus non-epitope protein sequences, implicating immune selection in driving strain diversification. Remarkably, CD4+ T-cell epitopes in EBNA2, EBNA-LP, and the EBNA3 family appeared to be under negative selection pressure, hinting towards a beneficial role of immune responses against these latency type III antigens in virus biology. These findings validate this immunoinformatics approach for providing novel insight into immune targets and the intricate relationship of host defense and virus evolution that may also pertain to other pathogens.
MeSH term(s)	Algorithms ; Antigenic Variation ; Antigens, Viral/genetics ; Antigens, Viral/immunology ; CD4-Positive T-Lymphocytes/immunology ; CD4-Positive T-Lymphocytes/virology ; CD8-Positive T-Lymphocytes/immunology ; CD8-Positive T-Lymphocytes/virology ; Data Mining ; Databases, Genetic ; Epitopes, T-Lymphocyte/genetics ; Epitopes, T-Lymphocyte/immunology ; Genetic Heterogeneity ; Herpesvirus 4, Human/genetics ; Herpesvirus 4, Human/immunology ; Polymorphism, Genetic
Chemical Substances	Antigens, Viral ; Epitopes, T-Lymphocyte
Language	English
Publishing date	2021-12-16
Publishing country	Switzerland
Document type	Comparative Study ; Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2606827-8
ISSN	1664-3224 ; 1664-3224
ISSN (online)	1664-3224
ISSN	1664-3224
DOI	10.3389/fimmu.2021.796379
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Characterization of a library of 20 HBV-specific MHC class II-restricted T cell receptors.

Schreiber, Sophia / Honz, Melanie / Mamozai, Weeda / Kurktschiev, Peter / Schiemann, Matthias / Witter, Klaus / Moore, Eugene / Zielinski, Christina / Sette, Alessandro / Protzer, Ulrike / Wisskirchen, Karin

Molecular therapy. Methods & clinical development

2021 Volume 23, Page(s) 476–489

Abstract: ... ...

Abstract	CD4
Language	English
Publishing date	2021-10-29
Publishing country	United States
Document type	Journal Article
ZDB-ID	2872938-9
ISSN	2329-0501 ; 2329-0501
ISSN (online)	2329-0501
ISSN	2329-0501
DOI	10.1016/j.omtm.2021.10.012
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Article ; Online: Virus and autoantigen-specific CD4+ T cells are key effectors in a SCID mouse model of EBV-associated post-transplant lymphoproliferative disorders.

Linnerbauer, Stefanie / Behrends, Uta / Adhikary, Dinesh / Witter, Klaus / Bornkamm, Georg W / Mautner, Josef

PLoS pathogens

2014 Volume 10, Issue 5, Page(s) e1004068

Abstract: Polyclonal Epstein-Barr virus (EBV)-infected B cell line (lymphoblastoid cell lines; LCL)-stimulated T-cell preparations have been successfully used to treat EBV-positive post-transplant lymphoproliferative disorders (PTLD) in transplant recipients, but ... ...

Abstract	Polyclonal Epstein-Barr virus (EBV)-infected B cell line (lymphoblastoid cell lines; LCL)-stimulated T-cell preparations have been successfully used to treat EBV-positive post-transplant lymphoproliferative disorders (PTLD) in transplant recipients, but function and specificity of the CD4+ component are still poorly defined. Here, we assessed the tumor-protective potential of different CD4+ T-cell specificities in a PTLD-SCID mouse model. Injection of different virus-specific CD4+ T-cell clones showed that single specificities were capable of prolonging mouse survival and that the degree of tumor protection directly correlated with recognition of target cells in vitro. Surprisingly, some CD4+ T-cell clones promoted tumor development, suggesting that besides antigen recognition, still elusive functional differences exist among virus-specific T cells. Of several EBV-specific CD4+ T-cell clones tested, those directed against virion antigens proved most tumor-protective. However, enriching these specificities in LCL-stimulated preparations conferred no additional survival benefit. Instead, CD4+ T cells specific for unknown, probably self-antigens were identified as principal antitumoral effectors in LCL-stimulated T-cell lines. These results indicate that virion and still unidentified cellular antigens are crucial targets of the CD4+ T-cell response in this preclinical PTLD-model and that enriching the corresponding T-cell specificities in therapeutic preparations may enhance their clinical efficacy. Moreover, the expression in several EBV-negative B-cell lymphoma cell lines implies that these putative autoantigen(s) might also qualify as targets for T-cell-based immunotherapy of virus-negative B cell malignancies.
MeSH term(s)	Animals ; Autoantigens/immunology ; CD4-Positive T-Lymphocytes/physiology ; Cells, Cultured ; Disease Models, Animal ; Epstein-Barr Virus Infections/immunology ; Epstein-Barr Virus Infections/transmission ; Herpesvirus 4, Human/immunology ; Herpesvirus 4, Human/physiology ; Humans ; Lymphoproliferative Disorders/immunology ; Lymphoproliferative Disorders/virology ; Mice ; Mice, Inbred C57BL ; Mice, SCID ; Peripheral Blood Stem Cell Transplantation/adverse effects ; Postoperative Complications/immunology ; Transplantation, Homologous/adverse effects
Chemical Substances	Autoantigens
Language	English
Publishing date	2014-05-22
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2205412-1
ISSN	1553-7374 ; 1553-7366
ISSN (online)	1553-7374
ISSN	1553-7366
DOI	10.1371/journal.ppat.1004068
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Article: Dynamic changes in circulating T follicular helper cell composition predict neutralising antibody responses after yellow fever vaccination.

Huber, Johanna E / Ahlfeld, Julia / Scheck, Magdalena K / Zaucha, Magdalena / Witter, Klaus / Lehmann, Lisa / Karimzadeh, Hadi / Pritsch, Michael / Hoelscher, Michael / von Sonnenburg, Frank / Dick, Andrea / Barba-Spaeth, Giovanna / Krug, Anne B / Rothenfußer, Simon / Baumjohann, Dirk

Clinical & translational immunology

2020 Volume 9, Issue 5, Page(s) e1129

Abstract: Objectives: T follicular helper (Tfh) cells are the principal T helper cell subset that provides help to B cells for potent antibody responses against various pathogens. In this study, we took advantage of the live-attenuated yellow fever virus (YFV) ... ...

Abstract	Objectives: T follicular helper (Tfh) cells are the principal T helper cell subset that provides help to B cells for potent antibody responses against various pathogens. In this study, we took advantage of the live-attenuated yellow fever virus (YFV) vaccine strain, YF-17D, as a model system for studying human antiviral immune responses Methods: We tracked and analysed the response of cTfh and other T and B cell subsets in peripheral blood of healthy volunteers by flow cytometry over the course of 4 weeks after YF-17D vaccination. Results: Using surface staining of cell activation markers to track YFV-specific T cells, we found increasing cTfh cell frequencies starting at day 3 and peaking around 2 weeks after YF-17D vaccination. This kinetic was confirmed in a subgroup of donors using MHC multimer staining for four known MHC class II epitopes of YF-17D. The subset composition of cTfh cells changed dynamically during the course of the immune response and was dominated by the cTfh1-polarised subpopulation. Importantly, frequencies of cTfh1 cells correlated with the strength of the neutralising antibody response, whereas frequencies of cTfh17 cells were inversely correlated. Conclusion: In summary, we describe detailed cTfh kinetics during YF-17D vaccination. Our results suggest that cTfh expansion and polarisation can serve as a prognostic marker for vaccine success. These insights may be leveraged in the future to improve current vaccine design and strategies.
Keywords	covid19
Language	English
Publishing date	2020-05-13
Publishing country	Australia
Document type	Journal Article
ZDB-ID	2694482-0
ISSN	2050-0068
ISSN	2050-0068
DOI	10.1002/cti2.1129
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Article ; Online: Epstein-Barr virus strain heterogeneity impairs human T-cell immunity.

Cirac, Ana / Stützle, Simon / Dieckmeyer, Michael / Adhikary, Dinesh / Moosmann, Andreas / Körber, Nina / Bauer, Tanja / Witter, Klaus / Delecluse, Henri-Jacques / Behrends, Uta / Mautner, Josef

Cancer immunology, immunotherapy : CII

2018 Volume 67, Issue 4, Page(s) 663–674

Abstract: The Epstein-Barr virus (EBV) establishes lifelong infections in > 90% of the human population. Although contained as asymptomatic infection by the immune system in most individuals, EBV is associated with the pathogenesis of approximately 1.5% of all ... ...

Abstract	The Epstein-Barr virus (EBV) establishes lifelong infections in > 90% of the human population. Although contained as asymptomatic infection by the immune system in most individuals, EBV is associated with the pathogenesis of approximately 1.5% of all cancers in humans. Some of these EBV-associated tumors have been successfully treated by the infusion of virus-specific T-cell lines. Recent sequence analyses of a large number of viral isolates suggested that distinct EBV strains have evolved in different parts of the world. Here, we assessed the impact of such sequence variations on EBV-specific T-cell immunity. With the exceptions of EBNA2 and the EBNA3 family of proteins, an overall low protein sequence disparity of about 1% was noted between Asian viral isolates, including the newly characterized M81 strain, and the prototypic EBV type 1 and type 2 strains. However, when T-cell epitopes including their flanking regions were compared, a substantial proportion was found to be polymorphic in different EBV strains. Importantly, CD4+ and CD8+ T-cell clones specific for viral epitopes from one strain often showed diminished recognition of the corresponding epitopes in other strains. In addition, T-cell recognition of a conserved epitope was affected by amino acid exchanges within the epitope flanking region. Moreover, the CD8+ T-cell response against polymorphic epitopes varied between donors and often ignored antigen variants. These results demonstrate that viral strain heterogeneity may impair antiviral T-cell immunity and suggest that immunotherapeutic approaches against EBV should preferably target broad sets of conserved epitopes including their flanking regions.
MeSH term(s)	Antigens, Viral/genetics ; Antigens, Viral/immunology ; CD8-Positive T-Lymphocytes/immunology ; CD8-Positive T-Lymphocytes/virology ; Epitopes, T-Lymphocyte/genetics ; Epitopes, T-Lymphocyte/immunology ; Epstein-Barr Virus Infections/genetics ; Epstein-Barr Virus Infections/immunology ; Epstein-Barr Virus Infections/virology ; Genetic Variation ; Herpesvirus 4, Human/classification ; Herpesvirus 4, Human/genetics ; Herpesvirus 4, Human/immunology ; Humans
Chemical Substances	Antigens, Viral ; Epitopes, T-Lymphocyte
Language	English
Publishing date	2018-01-27
Publishing country	Germany
Document type	Journal Article
ZDB-ID	195342-4
ISSN	1432-0851 ; 0340-7004
ISSN (online)	1432-0851
ISSN	0340-7004
DOI	10.1007/s00262-018-2118-z
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Article: Combination of Complement-Dependent Cytotoxicity and Relative Fluorescent Quantification of HLA Length Polymorphisms Facilitates the Detection of a Loss of Heterozygosity.

Witter, Klaus / Reibke, Roland / Subklewe, Marion / Zahn, Robert / Kauke, Teresa / Spiekermann, Karsten / Spannagl, Michael / Tischer, Johanna / Hiddemann, Wolfgang / Dick, Andrea

Bone marrow research

2014 Volume 2014, Page(s) 541345

Abstract: Loss of heterozygosity (LOH) is a common event in malignant cells. In this work we introduce a new approach to identify patients with loss of heterozygosity in the HLA region either at first diagnosis or after HLA mismatched allogeneic HSCT. Diagnosis of ...

Abstract	Loss of heterozygosity (LOH) is a common event in malignant cells. In this work we introduce a new approach to identify patients with loss of heterozygosity in the HLA region either at first diagnosis or after HLA mismatched allogeneic HSCT. Diagnosis of LOH requires a high purity of recipient target cells. FACS is time consuming and also frequently prevented by rather nonspecific or unknown immune phenotype. The approach for recipient cell enrichment is based on HLA targeted complement-dependent cytotoxicity (CDC). Relative fluorescent quantification (RFQ) analysis of HLA intron length polymorphisms then allows analysis of HLA heterozygosity. The approach is exemplified in recent clinical cases illustrating the detection of an acquired allele loss. As illustrated in one case with DPB1, distinct HLA loci in donor and patient were sufficient for both proof of donor cell removal and evaluation of allele loss in the patient's leukemic cells. Results were confirmed using HLA-B RFQ analysis and leukemia-associated aberrant immunophenotype (LAIP) based cell sort. Both results confirmed suspected loss of HLA heterozygosity. Our approach complements or substitutes for FACS-based cell enrichment; hence it may be further developed as novel routine diagnostic tool. This allows rapid recipient cell purification and testing for loss of HLA heterozygosity before and after allogeneic HSCT in easily accessible peripheral blood samples.
Language	English
Publishing date	2014-04-03
Publishing country	Egypt
Document type	Journal Article
ZDB-ID	2623734-9
ISSN	2090-3006 ; 2090-2999 ; 2090-2999
ISSN (online)	2090-3006 ; 2090-2999
ISSN	2090-2999
DOI	10.1155/2014/541345
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Article ; Online: Isolation and functional characterization of hepatitis B virus-specific T-cell receptors as new tools for experimental and clinical use.

Wisskirchen, Karin / Metzger, Kai / Schreiber, Sophia / Asen, Theresa / Weigand, Luise / Dargel, Christina / Witter, Klaus / Kieback, Elisa / Sprinzl, Martin F / Uckert, Wolfgang / Schiemann, Matthias / Busch, Dirk H / Krackhardt, Angela M / Protzer, Ulrike

PloS one

2017 Volume 12, Issue 8, Page(s) e0182936

Abstract: T-cell therapy of chronic hepatitis B is a novel approach to restore antiviral T-cell immunity and cure the infection. We aimed at identifying T-cell receptors (TCR) with high functional avidity that have the potential to be used for adoptive T-cell ... ...

Abstract	T-cell therapy of chronic hepatitis B is a novel approach to restore antiviral T-cell immunity and cure the infection. We aimed at identifying T-cell receptors (TCR) with high functional avidity that have the potential to be used for adoptive T-cell therapy. To this end, we cloned HLA-A02-restricted, hepatitis B virus (HBV)-specific T cells from patients with acute or resolved HBV infection. We isolated 11 envelope- or core-specific TCRs and evaluated them in comprehensive functional analyses. T cells were genetically modified by retroviral transduction to express HBV-specific TCRs. CD8+ as well as CD4+ T cells became effector T cells recognizing even picomolar concentrations of cognate peptide. TCR-transduced T cells were polyfunctional, secreting the cytokines interferon gamma, tumor necrosis factor alpha and interleukin-2, and effectively killed hepatoma cells replicating HBV. Notably, our collection of HBV-specific TCRs recognized peptides derived from HBV genotypes A, B, C and D presented on different HLA-A02 subtypes common in areas with high HBV prevalence. When co-cultured with HBV-infected cells, TCR-transduced T cells rapidly reduced viral markers within two days. Our unique set of HBV-specific TCRs with different affinities represents an interesting tool for elucidating mechanisms of TCR-MHC interaction and dissecting specific anti-HBV mechanisms exerted by T cells. TCRs with high functional avidity might be suited to redirect T cells for adoptive T-cell therapy of chronic hepatitis B and HBV-induced hepatocellular carcinoma.
MeSH term(s)	CD4-Positive T-Lymphocytes/immunology ; CD8-Positive T-Lymphocytes/immunology ; Coculture Techniques ; Female ; HLA-A2 Antigen/immunology ; Hepatitis B/immunology ; Hepatitis B Antigens/immunology ; Hepatitis B virus/genetics ; Hepatitis B virus/immunology ; Humans ; Male ; Middle Aged ; Receptors, Antigen, T-Cell/immunology ; Receptors, Antigen, T-Cell/metabolism ; Viral Proteins/metabolism
Chemical Substances	HLA-A*02 antigen ; HLA-A2 Antigen ; Hepatitis B Antigens ; Receptors, Antigen, T-Cell ; Viral Proteins
Language	English
Publishing date	2017-08-08
Publishing country	United States
Document type	Journal Article
ISSN	1932-6203
ISSN (online)	1932-6203
DOI	10.1371/journal.pone.0182936
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Article ; Online: Recruitment of highly cytotoxic CD8

Wagner, Karolin I / Mateyka, Laura M / Jarosch, Sebastian / Grass, Vincent / Weber, Simone / Schober, Kilian / Hammel, Monika / Burrell, Teresa / Kalali, Behnam / Poppert, Holger / Beyer, Henriette / Schambeck, Sophia / Holdenrieder, Stefan / Strötges-Achatz, Andrea / Haselmann, Verena / Neumaier, Michael / Erber, Johanna / Priller, Alina / Yazici, Sarah /

Roggendorf, Hedwig / Odendahl, Marcus / Tonn, Torsten / Dick, Andrea / Witter, Klaus / Mijočević, Hrvoje / Protzer, Ulrike / Knolle, Percy A / Pichlmair, Andreas / Crowell, Claudia S / Gerhard, Markus / D'Ippolito, Elvira / Busch, Dirk H

Cell reports

2021 Volume 38, Issue 2, Page(s) 110214

Abstract: T cell immunity is crucial for control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and has been studied widely on a quantitative level. However, the quality of responses, in particular of ... ...

Abstract	T cell immunity is crucial for control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and has been studied widely on a quantitative level. However, the quality of responses, in particular of CD8
MeSH term(s)	Adult ; CD8-Positive T-Lymphocytes/immunology ; COVID-19/immunology ; Cells, Cultured ; Cross Reactions/immunology ; Epitopes, T-Lymphocyte/immunology ; Female ; Humans ; Immunodominant Epitopes/immunology ; Leukocytes, Mononuclear/immunology ; Leukocytes, Mononuclear/virology ; Male ; Receptors, Antigen, T-Cell/immunology ; SARS-CoV-2/immunology ; Spike Glycoprotein, Coronavirus/immunology ; T-Lymphocytes, Cytotoxic/immunology
Chemical Substances	Epitopes, T-Lymphocyte ; Immunodominant Epitopes ; Receptors, Antigen, T-Cell ; Spike Glycoprotein, Coronavirus
Language	English
Publishing date	2021-12-17
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2649101-1
ISSN	2211-1247 ; 2211-1247
ISSN (online)	2211-1247
ISSN	2211-1247
DOI	10.1016/j.celrep.2021.110214
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Article ; Online: Single-cell RNA sequencing reveals ex vivo signatures of SARS-CoV-2-reactive T cells through 'reverse phenotyping'.

Fischer, David S / Ansari, Meshal / Wagner, Karolin I / Jarosch, Sebastian / Huang, Yiqi / Mayr, Christoph H / Strunz, Maximilian / Lang, Niklas J / D'Ippolito, Elvira / Hammel, Monika / Mateyka, Laura / Weber, Simone / Wolff, Lisa S / Witter, Klaus / Fernandez, Isis E / Leuschner, Gabriela / Milger, Katrin / Frankenberger, Marion / Nowak, Lorenz /

Heinig-Menhard, Katharina / Koch, Ina / Stoleriu, Mircea G / Hilgendorff, Anne / Behr, Jürgen / Pichlmair, Andreas / Schubert, Benjamin / Theis, Fabian J / Busch, Dirk H / Schiller, Herbert B / Schober, Kilian

Nature communications

2021 Volume 12, Issue 1, Page(s) 4515

Abstract: The in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly ... ...

Abstract	The in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we use single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induce transcriptional shifts by antigenic stimulation in vitro and take advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for 'reverse phenotyping'. This allows identification of SARS-CoV-2-reactive TCRs and reveals phenotypic effects introduced by antigen-specific stimulation. We characterize transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and show correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.
MeSH term(s)	Aged ; Aged, 80 and over ; CD4-Positive T-Lymphocytes/metabolism ; CD4-Positive T-Lymphocytes/virology ; COVID-19/epidemiology ; COVID-19/immunology ; COVID-19/virology ; Cells, Cultured ; Cohort Studies ; Female ; Gene Expression Profiling/methods ; Humans ; Male ; Middle Aged ; Pandemics ; Receptors, Antigen, T-Cell/genetics ; Receptors, Antigen, T-Cell/immunology ; Receptors, Antigen, T-Cell/metabolism ; SARS-CoV-2/physiology ; Sequence Analysis, RNA/methods ; Single-Cell Analysis/methods ; T-Lymphocytes/metabolism ; T-Lymphocytes/virology
Chemical Substances	Receptors, Antigen, T-Cell
Language	English
Publishing date	2021-07-26
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-021-24730-4
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