Upregulation of thioredoxin-1 in activated human NK cells confers increased tolerance to oxidative stress

Abstract

Adoptive transfer of immune cells, such as T lymphocytes and NK cells, has potential to control cancer growth. However, this can be counteracted by immune escape mechanisms within the tumor microenvironment, including those mediated by reactive oxygen species (ROS). Here, we determined the levels of anti-oxidant molecules in NK cells and their capacity to overcome ROS-induced immune suppression. We investigated the effect of H₂O₂ on resting NK cells, IL-2-activated NK cells and NK cells expanded by coculture with the K562 leukemia cell line genetically modified to express membrane-bound IL-15 and 4-1BB ligand (K562-mb15-41BBL). Expression of anti-oxidant and anti-apoptotic genes was evaluated by expression array, and protein levels of anti-oxidant molecules by Western blot. Activated NK cells, IL-2-activated NK cells and NK cells expanded by K562-mb15-41BBL were significantly more resistant to H₂O₂-induced cell death than resting NK. Thioredoxin-1 (TXN1) and peroxiredoxin-1 (PRDX1) were also up-regulated in activated NK cells. Moreover, H₂O₂-induced cell death after IL-2 activation was significantly induced in the presence of an anti-TXN1-neutralising antibody. Collectively, these data document that activated NK cells can resist to H₂O₂-induced cell death by up-regulation of TXN1.

Keywords: Hydrogen Peroxide; IL-2; Immune surveillance; NK cells; Thioredoxin-1.

Fig. 1

Apoptosis of NK cells after treatment with H₂O₂. a Purified NK cells were incubated with H₂O₂ for 24 h at indicated doses within physiological levels, and then subjected to apoptosis analysis with 7-AAD staining in combination with CD56 mAb. We prepared resting NK, IL-2 NK (500 IU/mL of IL-2), and expanded NK from the same healthy donor. Representative flow cytometric data from the same healthy donor are shown. b Summarized data of the apoptosis analysis with 7-AAD staining in NK cells from different donors (n = 8) are shown. ***p < 0.001, **p < 0.01, *p < 0.05

Fig. 2

Gene expression array of resting NK, IL-2 NK and expanded NK. a Resting NK (n = 5) were evaluated immediately after immunomagnetic separation of CD56 + CD3-cells. IL-2 NK (n = 6) and expanded NK (n = 5) were evaluated after 7 days of coculture. For the IL-2 NK, two concentrations of IL-2 were used: 200 IU/mL (n = 3) and 6000 IU/mL (n = 3). A representative heat map showed three major clusters derived from the unsupervised hierarchical clustering analysis, namely resting NK, IL-2 NK, and expanded NK samples. IL-2 NK samples were in one cluster comprising of two IL-2 concentrations used (dark blue, 200 IU/mL; light blue, 6000 IU/mL). b A representative bar graph shows the gene expression of four anti-oxidant genes, namely TXN1, PRDX1, SOD1, and SOD2, selected from the microarray data. Comparisons were made for TXN1, PRDX1, SOD1 between each group of NK cells with expanded NK versus IL-2 NK; expanded NK versus resting NK; IL-2 NK versus resting NK. No comparison was made for SOD2 since the expression showed opposite pattern. ***p < 0.001, **p < 0.01, *p < 0.05

Fig. 3

Expression of anti-oxidant molecules in activated NK cells. a Erk, Akt and anti-oxidant molecules were evaluated in three populations of NK cells: resting NK, IL-2 NK stimulated at 100 IU/mL of IL-2 and expanded NK. b Evaluation of PRDX1 and TXN1 protein expressions were carried out in resting NK with two populations of expanded NK stimulated with or without 500 IU/mL of IL-2, respectively (left); and with four populations of IL-2 NK stimulated with 1, 10, 100, and 1000 IU/mL of IL-2, respectively (right). c The SOD1, SOD2, TXN1, and PRDX1 protein expressions were evaluated in IL-2 NK and expanded NK from four different donors. These observations were obtained from three independent experiments

Fig. 4

TXN1 is responsible for NK cell resistance to H₂O₂-induced cell death. a IL-2 NK stimulated at 100 IU/mL of IL-2 were treated with H₂O₂ for 24 h with anti-TXN1-neutralising antibody (1 mg/mL) or without (Control). Representative viability data of IL-2 NK from four independent experiments is shown. *p < 0.05. b TXN1 protein expression was evaluated in five populations of NK cells: resting NK; IL-2 NK stimulated at 10 or 1000 IU/mL of IL-2 with or without anti-TXN1-neutralising Ab antibody. These observations were obtained from three independent experiments

Fig. 5

Cytotoxic assay in NK cells treated with H₂O₂. Resting NK (n = 5) and expanded NK (n = 5) were treated with H₂O₂ (0 and 80 µM) for 24 h, and then subjected to cytotoxic assay against K562 cells in a calcein-release assay. E:T ratio, effector:K562 ratio. ***p < 0.001, **p < 0.01, *p < 0.05