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doi:10.22028/D291-39997
Titel: | Identification of molecular candidates which regulate calcium-dependent CD8+ T-cell cytotoxicity |
VerfasserIn: | Zöphel, Sylvia Schäfer, Gertrud Nazarieh, Maryam Konetzki, Verena Hoxha, Cora Meese, Eckart Hoth, Markus Helms, Volkhard Hamed, Mohamed Schwarz, Eva C. |
Sprache: | Englisch |
Titel: | Molecular Immunology |
Bandnummer: | 157 |
Seiten: | 202-213 |
Verlag/Plattform: | Elsevier |
Erscheinungsjahr: | 2023 |
Freie Schlagwörter: | Calcium CTL (cytotoxic T lymphocytes) Real-time killing assay Cytotoxic efficiency Differential expression analyses Transcriptome data analyses |
DDC-Sachgruppe: | 500 Naturwissenschaften |
Dokumenttyp: | Journalartikel / Zeitschriftenartikel |
Abstract: | Cytotoxic CD8+ T lymphocytes (CTL) eliminate infected cells or transformed tumor cells by releasing perforincontaining cytotoxic granules at the immunological synapse. The secretion of such granules depends on Ca2+- influx through store operated Ca2+ channels, formed by STIM (stromal interaction molecule)-activated Orai proteins. Whereas molecular mechanisms of the secretion machinery are well understood, much less is known about the molecular machinery that regulates the efficiency of Ca2+-dependent target cell killing. CTL killing efficiency is of high interest considering the number of studies on CD8+ T lymphocytes modified for clinical use. Here, we isolated total RNA from primary human cells: natural killer (NK) cells, non-stimulated CD8+ T-cells, and from Staphylococcus aureus enterotoxin A (SEA) stimulated CD8+ T-cells (SEA-CTL) and conducted whole genome expression profiling by microarray experiments. Based on differential expression analysis of the transcriptome data and analysis of master regulator genes, we identified 31 candidates which potentially regulate Ca2+-homeostasis in CTL. To investigate a putative function of these candidates in CTL cytotoxicity, we transfected either SEA-stimulated CTL (SEA-CTL) or antigen specific CD8+ T-cell clones (CTL-MART-1) with siRNAs specific against the identified candidates and analyzed the killing capacity using a real-time killing assay. In addition, we complemented the analysis by studying the effect of inhibitory substances acting on the candidate proteins if available. Finally, to unmask their involvement in Ca2+ dependent cytotoxicity, candidates were also analyzed under Ca2+-limiting conditions. Overall, we identified four hits, CCR5 (C-C chemokine receptor type five), KCNN4 (potassium calcium-activated channel subfamily N), RCAN3 (regulator of calcineurin) and BCL (Bcell lymphoma) 2 which clearly affect the efficiency of Ca2+ dependent cytotoxicity in CTL-MART-1 cells, CCR5, BCL2, and KCNN4 in a positive manner, and RCAN3 in a negative way. |
DOI der Erstveröffentlichung: | 10.1016/j.molimm.2023.04.002 |
URL der Erstveröffentlichung: | https://doi.org/10.1016/j.molimm.2023.04.002 |
Link zu diesem Datensatz: | urn:nbn:de:bsz:291--ds-399975 hdl:20.500.11880/36010 http://dx.doi.org/10.22028/D291-39997 |
ISSN: | 0161-5890 |
Datum des Eintrags: | 21-Jun-2023 |
Bezeichnung des in Beziehung stehenden Objekts: | Supporting information |
In Beziehung stehendes Objekt: | https://ars.els-cdn.com/content/image/1-s2.0-S0161589023000779-mmc1.docx |
Fakultät: | M - Medizinische Fakultät NT - Naturwissenschaftlich- Technische Fakultät |
Fachrichtung: | M - Biophysik M - Humangenetik NT - Biowissenschaften |
Professur: | M - Prof. Dr. Markus Hoth M - Prof. Dr. Eckhart Meese NT - Prof. Dr. Volkhard Helms |
Sammlung: | SciDok - Der Wissenschaftsserver der Universität des Saarlandes |
Dateien zu diesem Datensatz:
Datei | Beschreibung | Größe | Format | |
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1-s2.0-S0161589023000779-main.pdf | 3,38 MB | Adobe PDF | Öffnen/Anzeigen |
Diese Ressource wurde unter folgender Copyright-Bestimmung veröffentlicht: Lizenz von Creative Commons