Abstract
Interleukin 15 (IL-15) is one of the most important cytokines that regulate the biology of natural killer (NK) cells1. Here we identified a signaling pathway—involving the serine-threonine kinase AKT and the transcription factor XBP1s, which regulates unfolded protein response genes2,3—that was activated in response to IL-15 in human NK cells. IL-15 induced the phosphorylation of AKT, which led to the deubiquitination, increased stability and nuclear accumulation of XBP1s protein. XBP1s bound to and recruited the transcription factor T-BET to the gene encoding granzyme B, leading to increased transcription. XBP1s positively regulated the cytolytic activity of NK cells against leukemia cells and was also required for IL-15-mediated NK cell survival through an anti-apoptotic mechanism. Thus, the newly identified IL-15–AKT–XBP1s signaling pathway contributes to enhanced effector functions and survival of human NK cells.
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All summary or representative data generated and supporting the findings of this study are available within the paper. Raw data that support the findings of this study are available upon reasonable request.
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Acknowledgements
This work was supported by grants from the National Institutes of Health (AI129582 and NS106170 to J.Y. and CA185301, CA210087 and CA068458 to M.A.C.), the Leukemia and Lymphoma Society (6503-17 and 1364-19 to J.Y.), the American Cancer Society (RSG-14-243-01-LIB to J.Y.) and the Gabrielle’s Angel Cancer Research Foundation (#87 to J.Y.).
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Y.W. performed experiments, designed research and wrote the manuscript; Y.Z., P.Y., W.D., Z.Z. and L.C. performed experiments; A.P.N. revised and proofread the manuscript; J.Z. performed statistical analyses; D.M.B., B.L.M-B., A.G.F. and M.A.C. designed research, reviewed the manuscript and/or acquired funding; J.Y. conceptualized the idea, designed research, wrote the manuscript, acquired funding and supervised the study.
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Supplementary Figure 1 Quantification of IL-15-induced XBP1s and its correlation with NK cell cytotoxicity.
a, Densitometric quantification of the level of XBP1s protein normalized to the level of β-Actin protein, assessed by immunoblotting shown in Fig. 1b. NK cells were treated with IL-2 (100 units/ml) or IL-15 (100 units/ml) for 24 h. Immunoblotting was performed with an anti-XBP1s antibody. Bar graphs display mean ± s.d. of n = 8 donors. ***P < 0.001 by linear mixed model. b, NK cells were treated with IL-15 (100 units/ml) for 24 h and co-cultured with MM.1S multiple myeloma cells for 4 h. CD107a surface expression and intracellular level of XBP1s were determined by flow cytometry using anti-CD107a and anti-XBP1s antibodies. n = 4 donors. ***P < 0.001 by two-tailed paired t test. MFI, mean fluorescence intensity.
Supplementary Figure 2 XBP1s positively regulates the expression of GZMB in primary NK cells.
a,b, NK cells were pre-treated with 1 µM of thapsigargin (Thap) for 1 h and then treated with or without IL-15 (100 units ml−1) for 4 h followed by qPCR (a) or for 24 h followed by immunoblotting to detect XBP1s or GZMB expression (b). Bar graphs display mean ± s.e.m. of n = 3 donors. *P < 0.05, **P < 0.01, ***P < 0.001 by linear mixed model (a). c, Densitometric quantification of the level of XBP1s or GZMB protein normalized to the level of β-Actin protein in (b). Bar graphs display mean ± s.d. of n = 3 donors. **P < 0.01 by linear mixed model. d, NK cells were transfected with XBP1 or a scramble siRNA by electroporation and then were cultured for 48 h, followed by immunoblotting with XBP1s, GZMB, and β-Actin antibodies. e, Densitometric quantification of the level of XBP1s or GZMB protein normalized to the level of β-Actin protein in (d). Bar graphs display mean ± s.d. of n = 3 donors. **P < 0.01, ***P < 0.001 by Student’s two-tailed unpaired t test. f,g,h, NK cells were pretreated with 50 µM of 4µ8C for 1 h and then treated with or without IL-15 (100 units ml−1) for 4 h followed by qPCR (f) or for 24 h followed by immunoblotting to detect XBP1s or GZMB expression (g,h). h, Densitometric quantification of the level of XBP1s or GZMB protein normalized to the level of β-Actin protein in (g). Bar graphs display mean ± s.e.m. (f) or mean ± s.d. (h) of n = 3 donors. **P < 0.01, ***P < 0.001 by linear mixed model. The experiment was repeated independently for three donors with similar results (b,d,g). N.S., no significance. Blot images (b,d,g) were cropped, and the full scans are shown in the supplementary figures.
Supplementary Figure 3 Interaction and co-localization of XBP1s with T-BET.
a, Densitometric quantification of the level of T-BET protein normalized to input (i.e., enrichment) in Fig. 3c. 293T cells were co-transfected with T-BET and FLAG-XBP1u, FLAG-XBP1s, or the control (pCDH) lentiviral vector by lentiviral infection, followed by a 48 h culture. Co-IP was performed with an anti-FLAG antibody. Bar graphs display mean ± s.d. of n = 3 independent experiments. **P < 0.01, ***P < 0.001 by linear mixed model. b, NK-92 cells were transduced with FLAG-XBP1s or the control (pCDH) lentiviral vector by lentiviral infection, followed by a 24 h culture. Immunofluorescent staining was performed with anti-FLAG and anti-T-BET antibodies. The nuclei were stained by DAPI. Yellow indicates overlap of red and green. c, NK-92 (upper panel) or primary NK cells (lower panel) were treated with IL-15 (100 units/ml) for 24 h, followed by immunofluorescent staining with an anti-T-BET antibody that was different from the antibody clone used in (b) and Fig. 3d for data validation. An anti-α-tubulin antibody was included to stain the cytoplasm. The nuclei were stained by DAPI. The experiment was repeated independently for one (b) or three (c) times with similar results. d, Primary human NK cells were treated with or without 50 μM of 4μ8c in the presence of IL-15 (100 units/ml) for 16 h. ChIP was performed with a T-BET antibody or control IgG. Precipitated DNA was then analyzed by PCR. Bar graphs display mean ± s.d. of n = 4 donors. **P < 0.01 by Student’s two-tailed unpaired t test.
Supplementary Figure 4 XBP1s positively regulates GZMB gene promoter activity in a STAT5-independent manner.
a,b, 293T cells were co-transfected with the following four plasmids: 1. STAT5A shRNA, STAT5B shRNA, or scramble shRNA; 2. XBP1s or empty vector pCDH (EV); 3. A pGL3 plasmid containing the GZMB promoter; 4. A pRL-TK plasmid (as a control for data normalization). The transfected cells were cultured for 48 h and then harvested and lysed to determine the expression level of XBP1s or STAT5 protein by immunoblotting (a) or GZMB promoter activity by luciferase reporter assays (b). Bar graphs display mean ± s.d. of n = 4 independent experiments. ***P < 0.001 by Student’s two-tailed unpaired t test. The moderate knockdown effect for STAT5A is likely because the antibody used (#25656, Cell Signaling Technology, Inc.) recognizes both STAT5A and STAT5B. N.S., no significance. The experiment in (a) was repeated independently for three times with similar results. Blot images (a) were cropped, and the full scans are shown in the supplementary figures.
Supplementary Figure 5 AKT signaling regulates the expressions of XBP1s and GZMB.
a, Densitometric quantification of the level of XBP1s protein normalized to the level of β-actin protein in Fig. 5c. NK cells were pre-treated with or without 10 µM of AKTi-1/2, a potent isozyme selective Akt1/2 kinase inhibitor, followed by stimulation with IL-15 (100 units ml−1) for 6 h. Cells were then harvested to make lysates for immunoblot analysis. Bar graphs display mean ± s.d. of n = 3 donors. *P < 0.05 by linear mixed model. b,c, AKT1 or scramble shRNA lentiviral construct (pLKO.1) was transduced by lentiviral infection in primary human NK cells for 24 h, followed by fluorescence-activated cell sorting (FACS) for transduced cells based on GFP expression. GZMB expression was analyzed at the protein level by flow cytometry (b) or at the mRNA level by qPCR (c). Bar graphs display mean ± s.d. in (b) or mean ± s.e.m. in (c) of n = 3 donors. **P < 0.01, ***P < 0.001 by Student’s two-tailed paired t test. d,e, myrAKTΔ4-129 lentiviral construct or empty vector (EV) was transduced by lentiviral infection in human primary NK cells for 24 h, followed by FACS-based cell sorting for GFP(+) cells. GZMB expression of the sorted cells was analyzed as described in (b, c). Bar graphs display mean ± s.d. in (d) or mean ± s.e.m. in (e) of n = 3 donors. P = 0.07, **P < 0.01, ***P < 0.001 by Student’s two-tailed paired t test. N.S., no significance.
Supplementary Figure 6 AKT signaling regulates XBP1s at the protein level.
a, Densitometric quantification of the level of XBP1s protein normalized to the level of β-actin in Fig. 5d. NK cells were pre-treated with IL-15 (100 units/ml) alone or IL-15 plus 10 µM of AKTi-1/2 for 1 h; then cells were cultured with or without 10 µg/ml of cycloheximide (CHX) for 30 min without washing cells. Cells were then harvested to make lysates for immunoblot analysis. Bar graphs display mean ± s.d. of n = 3 donors. P = 0.08, **P < 0.01 by linear mixed model. b, Densitometric quantification of the level of XBP1s protein normalized to the level of β-actin protein in Fig. 5e. 293T cells were co-transfected with 0.04, 0.2, or 1 µg of pECE-myrAKTΔ4-129 vector or pECE empty vector (EV) and pCDH-FLAG-tagged XBP1s (FLAG-XBP1s) vector or pCDH control vector. The transfected cells were cultured for 24 h, followed by detecting XBP1s protein expression by immunoblotting with an anti-XBP1s antibody. Bar graphs display mean ± s.d. of n = 3 independent experiments. P = 0.06, *P < 0.05, **P < 0.01 by linear mixed model. c, A plasmid containing myrAKTΔ4-129 with constitutively active AKT or a control plasmid was co-transfected with an XBP1s plasmid, a pGL3 plasmid containing the GZMB promoter, and a pRL-TK plasmid (as a control for data normalization) into 293T cells. The transfected cells were cultured for 48 h. The promoter activity of the GZMB gene was detected by luciferase reporter assays and the expression of XBP1s and myrAKTΔ4-129 at the protein levels were detected by immunoblot. Bar graphs display mean ± s.d. of n = 3 independent experiments. ***P < 0.001 by two-way ANOVA model. The blot experiment was repeated independently for three times with similar results. Blot images (c) were cropped, and the full scans are shown in the Supplementary Figures. d, Densitometric quantification of the level of XBP1s protein normalized to the level of β-actin protein in Fig. 5g. 293T cells were co-transfected with a pECE-myrAKTΔ4-129 vector or pECE empty vector (EV) and pCDH-FLAG-tagged XBP1s (FLAG-XBP1s) vector or pCDH control vector, followed by treatment with 10 µg/ml of CHX for 0, 1, 2 and 4 h in the presence or absence of 10 µM of MG132, a protein degradation inhibitor. Bar graphs display mean ± s.d. of n = 3 independent experiments. **P < 0.01, ***P < 0.001 by linear mixed model. N.S., no significance. Blot images (c) were cropped, and the full scans are shown in Supplementary Figures.
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Wang, Y., Zhang, Y., Yi, P. et al. The IL-15–AKT–XBP1s signaling pathway contributes to effector functions and survival in human NK cells. Nat Immunol 20, 10–17 (2019). https://doi.org/10.1038/s41590-018-0265-1
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DOI: https://doi.org/10.1038/s41590-018-0265-1
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