TBKBP1 overexpression in restimulated CD8<sup>+</sup> T cells results in increased TBK1 phosphorylation.
<p>PBMCs obtained from CMV-seronegative healthy donors were stimulated with plate-bound anti-human CD3 and anti-human CD28 for 48 hours and subsequently retrovirally transduced with TBKBP1-expressing vectors or empty vector (EV) controls. Successfully transduced CD8<sup>+</sup>mChe...
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2024
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| Summary: | <p>PBMCs obtained from CMV-seronegative healthy donors were stimulated with plate-bound anti-human CD3 and anti-human CD28 for 48 hours and subsequently retrovirally transduced with TBKBP1-expressing vectors or empty vector (EV) controls. Successfully transduced CD8<sup>+</sup>mCherry<sup>+</sup> T cells were sorted by flow cytometry, serum-starved overnight, and subsequently stimulated with soluble anti-human CD3, anti-human CD28, and cross-linking antibodies, while unstimulated cells served as additional controls. Samples from both unstimulated (-) and stimulated (+) EV-transduced or TBKBP1-overexpressing CD8<sup>+</sup> T cells were subjected to immunoblotting to determine the expression of <b>(A)</b> TBKBP1, <b>(B)</b> TBK1, and <b>(C)</b> phosphorylated TBK1 (pTBK1). The analysis of GAPDH expression served as loading control. <b>(D)</b> Band intensities for pTBK1 and TBK1 were quantified and the fold change of TBK1 phosphorylation upon stimulation of EV-transduced or TBKBP1-overexpressing CD8<sup>+</sup> T cells was determined by dividing the ratio of pTBK1/TBK1 band intensities of stimulated cells to the ratio of pTBK1/TBK1 band intensities of unstimulated samples. Data are combined from 4 independent donors analysed in 2 independent experiments. <b>(E)</b> EV-transduced or TBKBP1-overexpressing CD8<sup>+</sup> T cells were stimulated in the presence of a PKCθ inhibitor (+) or treated with DMSO as control (-). Subsequently, CD3<sup>+</sup>CD8<sup>+</sup> cells were analysed for intracellular IFN-γ expression by flow cytometry. The geometric mean fluorescent intensity (gMFI) of IFN-γ expression among CD3<sup>+</sup>CD8<sup>+</sup> cells is depicted. Data are combined from 4 independent donors analysed in 2 independent experiments. <b>(D, E)</b> For statistical analyses, a paired two-tailed student’s <i>t</i> test was conducted with *, p ≤ 0.05.</p> |
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