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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

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