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. 2020 Nov 23;10:20363. doi: 10.1038/s41598-020-76702-1

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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OC-expanded NK cells from cancer patients have much lower capacity to expand, mediate cytotoxicity, and secrete IFN-γ. OCs were generated as described in Materials and Methods. NK cells (1 × 10⁶ cells/ml) from healthy individuals and cancer patients were treated with a combination of IL-2 (1000 U/ml) and anti-CD16mAb (3 µg/ml) for 18 h before they were cultured with the healthy individuals’ OCs and sAJ2 at a ratio of 1:2:4 (OCs:NK:sAJ2). On days 6, 9, 12, and 15 of co-culture, the numbers of lymphocytes were counted using microscopy (n = 70) (A). NK cells were treated and cultured as described in Fig. 1A. Cytotoxicity of day 15 cultured NK cells was determined using standard 4-h ⁵¹Cr release assay against OSCSCs. The Lytic units (LU) 30/10⁶ cells were determined using the inverse number of NK cells required to lyse 30% of OSCSCs × 100 (n = 16) (B). NK cells were treated and cultured as described in Fig. 1A. On days 6, 9, 12, and 15, supernatants were harvested from the co-cultures to determine IFN-γ secretion using single ELISA (n = 63) (C). The amounts of IFN-γ secretion shown in Fig. 1C were determined based on 1 × 10⁶ cells (n = 63) (D). T cells (1 × 10⁶ cells/ml) from healthy individuals and cancer patients were treated with a combination of IL-2 (100 U/ml) and anti-CD3 (1 µg/ml)/CD28mAb (3 µg/ml) for 18 h before they were co-cultured with healthy individuals’ OCs and sAJ2 at a ratio of 1:2:4 (OCs:T:sAJ2). On days 6, 9, 12, and 15, the T cells were counted using microscopy; the cumulative cell counts from day 0 to day 15 are displayed in the figure (n = 7) (E). T cells were treated and cultured as described in Fig. 1E. The supernatants were harvested on days 6, 9, 12, and 15, and the levels of IFN-γ secretion were determined using single ELISA (n = 42) (F). Amounts of IFN-γ secretion shown in Fig. 1F were assessed based on 1 × 10⁶ cells (n = 42) (G). NK cells and T cells were treated and cultured as described in Fig. 1A and Fig. 1E respectively. The cells were counted using microscopy on days 6, 9, 12, and 15; the cumulative cell counts from day 0 to day 15 are displayed in the figure (n = 10) (H). NK and T cells were treated and cultured as described in Fig. 1A and Fig. 1E respectively. The supernatants were then harvested on days 6, 9, 12, and 15 of the co-cultures and the amounts of IFN-γ secretion were determined using single ELISA; the cumulative levels of IFN-γ secretion from day 0 to day 15 are displayed in the figure (n = 10) (I).