Quantification of cytokines from the ARMATA.

<p>PBMCs isolated from healthy CMV-seronegative donors were stimulated with plate-bound anti-human CD3 and anti-human CD28 antibodies and subsequently co-transduced with mTCR and TBKBP1- or EV-mCherry plasmids. Successfully transduced CD8<sup>+</sup>mTCR<sup>+</sup>mCherry<sup>+</sup> T cells were sorted from both TBKBP1-overexpressing samples and EV-transduced controls using flow cytometry and co-cultured with CMV-infected MRC-5 cells followed by the ARMATA. 36 hours after infection, culture supernatants were harvested and cytokine profiles determined from cultures of CMV-infected MRC-5 cells without the addition of CD8<sup>+</sup> T cells (w/o T cells), in the presence of EV-transduced mTCR 5–2<sup>+</sup> CD8<sup>+</sup> T cells (EV) or in the presence of TBKBP1-overexpressing mTCR 5–2<sup>+</sup> CD8<sup>+</sup> T cells (TBKBP1) with E:T ratios of 0.5:1 (left) and 1:1 (right) for indicated cytokines. Data for IFN-γ, Granzyme A, Granzyme B and Perforin from the E:T ratio of 1:1 are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1012581#ppat.1012581.g007" target="_blank">Fig 7</a>. Data from 3 independent experiments with 2 technical replicates each are shown. Black dots indicate frequencies from individual donors and grey bar mean values with SD. For statistical analyses, a paired two-tailed student’s <i>t</i> test (leaving out “w/o T cell” group) was conducted with *, p ≤ 0.05 and **, p ≤ 0.01.</p> <p>(PDF)</p>