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. 2019 Jul 3;216(9):2113–2127. doi: 10.1084/jem.20181454

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© 2019 Prager et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).

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Figure 3. — NK cells differentially induce GrzB- or Casp8-mediated target cell death during serial killing. Serial killing activity of primary human NK cells was evaluated against HeLa-CD48 cells stably expressing NES-ELQTD-GFP-T2A-NES-VGPD-mCherry. (A–G) Three independent experiments using IL-2–activated NK cells from three donors (A–E) and three independent experiments using resting, freshly isolated NK cells from three different donors (F and G) were conducted. A total of 347 activated and 63 resting NK cells was tracked over 15–17 h, and each of their killing events was characterized in terms of GrzB and Casp8 activity as well as morphology of cell death. (A) Example of five consecutive killing events by a serial killer NK cell (NK cell visualized in brightfield, arrowheads, lower row) with corresponding enzymatic activity (red and green nuclear signal indicates GrzB and Casp8 activity, respectively). Images have been background-subtracted and adjusted for brightness and contrast using ImageJ. See Video 3 for details. Scale bar, 15 µm. (B and C) Diagrams display the activity of GrzB and Casp8 (B) and the morphology of target cell death (C) in single killing events. Each row displays the killing sequence of one individual NK cell. Cells for which fluorescent signal could not be reliably measured are denoted by “unstained.” Some target cells showed strong Casp8 reporter activity without displaying significant morphological signs of cell death during the observation period (No death). (D–G) Target cell deaths were categorized according to their respective number in the corresponding NK cell killing sequence. Histograms show the proportion of target cell deaths displaying activity of GrzB, Casp8, or absence of reporter cleavage (D and F) and the proportion of target cell deaths with an apoptosis-like or non-apoptosis-like phenotype (E and G). Cells showing clear enzymatic activity following an NK cell interaction, yet surviving the entire time span of the assay, are denoted by “No death.” The non-apoptosis-like phenotype is classified as described in Fig. 2, and the apoptosis-like fraction also contains cells with the “intermediate” phenotype.

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