Natural killer (NK) cells are critical mediators of host immunity to pathogens. Here, we demonstrate that the endoplasmic reticulum stress sensor inositol-requiring enzyme 1 (IRE1α) and its substrate transcription factor X-box-binding protein 1 (XBP1) drive NK cell responses against viral infection and tumors in vivo. IRE1α-XBP1 were essential for expansion of activated mouse and human NK cells and are situated downstream of the mammalian target of rapamycin signaling pathway. Transcriptome and chromatin immunoprecipitation analysis revealed c-Myc as a new and direct downstream target of XBP1 for regulation of NK cell proliferation. Genetic ablation or pharmaceutical blockade of IRE1α downregulated c-Myc, and NK cells with c-Myc haploinsufficency phenocopied IRE1α-XBP1 deficiency. c-Myc overexpression largely rescued the proliferation defect in IRE1α−/− NK cells. Like c-Myc, IRE1α-XBP1 also promotes oxidative phosphorylation in NK cells. Overall, our study identifies a IRE1α-XBP1-cMyc axis in NK cell immunity, providing insight into host protection against infection and cancer.
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The RNA-seq data were deposited in the Gene Expression Omnibus under the accession number GSE113214. The remaining data that support the findings of this study are available from the corresponding authors upon request. Materials will be provided with material transfer agreements in place as appropriate.
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We thank E. Vivier (Aix Marseille University) for Ncr1iCre mice; R. Wang (Nationwide Children’s Hospital) for Mycfl/fl mice; R. Sears (Oregon Health & Science University) for Mycfsf/fsf mice; J. Ritz (Dana-Farber Cancer Institute) for the NKL cell line; C. Hurley (Georgetown University) for the KHYG-1 cell line; D. Lyden (Weill Cornell Medicine) for the B16F10 cell line; members of the Sun laboratory for sharing viral reagents, cytokines and flow cytometry antibodies and for providing experimental assistance; members of the Glimcher laboratory—M. Raundhal, C. Lentucci, R. Xu, S. Ghosh and A. Gonzalez for technical support; S. Adoro (Case Western Reserve University) and J. Cubillos-Ruiz (Weill Cornell Medicine) for critical reading of the manuscript and helpful discussion; M. Song (Weill Cornell Medicine) and the Wucherpfennig laboratory (Dana-Farber Cancer Institute) for insightful comments; J. Xiang (Weill Cornell Genomics and Epigenomics Core) for RNA-seq; J. McCormick (Weill Cornell Medicine) and DFCI Jimmy Fund Flow Cytometry Core for cell sorting; Specialized Histopathology Core at Brigham & Women’s Hospital for histology; L. Cohen-Gould and J. Jimenez (Microscopy and Image Analysis Core Facility at Weill Cornell Medicine) for electron microscopy; X. Liu (Metabolism and Mitochondrial Research Core, Beth Israel Deaconess Medical Center) for metabolic flux assays; D. Neuberg (Dana-Farber Cancer Institute) for advising on statistical analysis; and S. Schneider (Dana-Farber Cancer Institute) for manuscript editing. L.H.G. and H.D. were supported by Institutional Funding from The Dana-Farber Cancer Institute. H.D. was also supported by a Helen Gurley Brown Presidential Initiative Award (Dana-Farber Cancer Institute). J.C.S. was supported by the Ludwig Center for Cancer Immunotherapy, the American Cancer Society, the Burroughs Wellcome Fund and the NIH (grant nos. AI100874, AI130043 and P30CA008748). N.M.A. was supported by an MSTP Grant from the National Institute of General Medical Sciences of the NIH to the Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD-PhD Program (grant no. T32GM007739) and by an F30 Predoctoral Fellowship from National Institue of Allergy and Infectious Diseases of the NIH (no. F30 AI136239). X.C. was supported by a NIH grant (no. 1 R37 CA228304-01).
L.H.G. is a former Director of Bristol-Myers Squibb and is currently on the board of directors of and holds equity in GlaxoSmithKline Pharmaceuticals and the Waters Corporation. She chairs the scientific advisory board, is a co-founder of and holds equity in Quentis Therapeutics. She also serves on the scientific advisory boards of Repare Therapeutics, Abpro and Kaleido Therapeutics.
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Integrated supplementary information
Supplementary Figure 1 qPCR validation of upregulation of the IRE1α/XBP1 pathway in activated NK cells, and requirement of STAT4 and mTOR signaling pathways in driving IRE1α /XBP1.
(a) Quantitative real time PCR analysis of indicated UPR genes in sorting, purified splenic NK cells from wild-type mice at day 1 post MCMV infection. β-ACTIN was used as reference, and data were normalized to the uninfected control expression levels. (b) Quantitative real time PCR analysis of indicated UPR genes in sorting-purified splenic NK cells from wild-type mice after 16 hr culture in the presence or absence of IL-12 and IL-18. (c) Heat map of RNA-seq analysis (GSE106138) showing the expression of canonical IRE1α/XBP1 target genes in WT and STAT4-deficient Ly49H+ NK cells sorted from the spleens of mixed BM chimeric mice at day 2 PI. (d) Flow cytometric analysis of IRE1α activation in cytokine-activated NK cells after pharmaceutical inhibition of mTOR. NK cells from ERAI reporter mice were stimulated with mouse recombinant IL-12 (20 ng/ml) and IL-18 (10 ng/ml) for 6 hrs in the presence of mTOR inhibitor rapamycin (Rapa, 5 nM and 10 nM for low and high dose, respectively), Ku-0063794 (Ku, 1.5 μM and 3 μM for low and high dose, respectively), PP242 (PP, 0.5 μM and 1 μM for low and high dose, respectively), or IRE1 inhibitor 4μ8C (2.5 μM and 5 μM for low and high dose, respectively), or DMSO control. Representative flow cytometric plot (upper) and quantifications of relative inhibition efficiency (bottom) are shown. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.0001 and **** p < 0.0001. Two-way analysis of variance (ANOVA) with the Sidak post-test performed on a. Two tailed unpaired Student’s t-test performed on b. All error bars, mean with s.d. from biological replicates. n = 3 mice/group in a. n = 3 mice in b; with technical triplicates for ex vivo culture. Each column is a different mouse in c. n = 3 ERAI mice in d. Experiments were independently repeated three (a, b) and two (d) times.
(a) Validation of IRE1NK KO efficiency: XBP1 splicing assay (upper panel) and quantitative real time PCR analysis (bottom panel) of indicated UPR genes in sorting-purified splenic NK cells from IRE1NK and IRE1f/f littermate mice after 4 hr ex vivo incubation in the presence or absence of tunicamycin, a pharmacologic inducer of ER stress. The quantitative real time PCR data are presented as relative expression to β-Actin. (b) Flow cytometric analysis of NK cell percentages and absolute numbers in BM, spleen and lung of IRE1NK and XBP1NK naïve mice, in comparison to their Cre-littermate controls. BM NK cells are identified by Lin−CD122 and splenic NK cells are identified by Lin−NK1.1+. (c) Flow cytometric analysis of NK cell development in BM of IRE1NK and XBP1NK mice: percentages of NK cells that are NK progenitors (NKP, DX5− NK1.1−), immature NK cells (iNK, DX5− NK1.1+) or mature NK (mNK, DX5− NK1.1+) are shown. (d) Flow cytometric analysis of NK cell maturation in spleen of IRE1NK and XBP1NK mice: percentages of NK cells that are immature (CD27+ CD11b−), mature (CD27− CD11b+) or intermediate stage (CD27+ CD11b+) are shown. (e) Flow cytometric analysis of NK cell repopulation in mixed BM chimera mice: WT (CD45.1): IRE1NK (CD45.2), and WT (CD45.1): IRE1vav1 (CD45.2) BM chimeras were generated as in the schematic (left); NK cell repopulation in the irradiated recipient mice was assessed in the peripheral blood at week 8 after BM transfer (right). (f) As in d, representative histogram plots showing surface expression of cytokine receptors and activating receptors in splenic NK cells from naïve IRE1NK and XBP1NK in comparison to their Cre- littermate control mice. ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 and ****p < 0.0001. Two tailed unpaired Student’s t-test performed on a. All error bars, mean with s.d.. n = 3 mice/group in a, f. n = 5 mice/group in b-d. n = 10 mice/group in e. Experiments were independently repeated three (a-e) and two (a, f) times.
Supplementary Figure 3 Dispensable function of IRE1α/XBP1 in NK cell priming, cytokine production or cytotoxicity.
(a) Gating strategy applied in analysis of co-transfer and mixed BM chimera experiments. (b-c) Flow cytometric analysis of splenic WT or IRE1NK Ly49H+ NK cells from mixed BM chimeric mice as indicated in Supplementary Figure 2e at day 2 PI: (b)IFN-γ and (c)CD69 and intracellular Granzyme B. (d) Flow cytometric analysis of co-transferred WT or IRE1NK Ly49H+ NK cells from the spleen of Ly49H-deficient recipients at day 2 PI: pan-Akt, p-Akt, and p-S6 with quantification normalized to WT expression levels as 100%. Two tailed unpaired Student’s t-test performed on b and c. All error bars, mean with s.d. n = 6 mixed BM chimera mice in b. n = 4 mice in c. n = 3 mice in d. Data were independently repeated three times.
Supplementary Figure 4 Minimal impact of IRE1α depletion on RIDD in infection-activated NK cells, and specific pharmaceutical blockade of IRE1α by 4μ8C.
(a) The volcano plot of RNA-seq analysis showing all genes in IRE1NK versus WT Ly49H+ splenic NK cells harvested from IRE1NK (CD45.2): WT (CD45.1) mixed BM chimera mice day 1.5 PI. RIDD target genes1 and XBP1 target genes1 are highlighted in red and blue, respectively. (b) IRE1NK and IRE1f/f NK cells from littermate animals were pre-labeled with CTV and cultured ex vivo with IL-2 and IL-15 as in Fig. 4f, g, but in the presence of 4μ8C (5 uM) or DMSO control treatment. Representative flow cytometric plots of CTV dilution and Ki-67 levels at day 3 are shown. Proliferating cells are defined as Ki-67+ CTVlo. n = 2 mice/group in b with technical duplicates per mouse. Experiments in b were independently repeated two times.
1. So, J.S. et al. Silencing of lipid metabolism genes through IRE1alpha-mediated mRNA decay lowers plasma lipids in mice. Cell Metab 16, 487-499 (2012).
Representative electron microscopy of IRE1NK versus WT Ly49H+ splenic NK cells harvested from IRE1NK(CD45.2): WT (CD45.1) mixed BM chimera mice day 7 PI. The high-resolution plots at bottom show mitochondrial morphology. n = 3 mixed BM chimera mice.
(a) IPA Upstream Analysis derived from RNA-seq (day 1.5 PI) as indicated in Fig. 6a: prediction of Myc regulation in IRE1NK cells. (b) IPA analysis of Functional overlap between IRE1- and c-Myc-regulated genes derived from RNA-seq analysis of Ly49H+ IRE1NK cells at day 1.5 PI. (c) Flow cytometric analysis of c-Myc induction at the level of transcription (upper row) and translation (middle row) in splenic NK cells from WT mice either naïve or at day 1 PI. (bottom row) Representative flow cytometric plots indicating the concomitant induction of c-Myc protein and Venus reporter expression in NK cells from indicated organs of ERAI transgenic mice at day 2 PI. (d) Quantitative real time PCR and flow cytometric analysis of the basal levels of c-Myc mRNA and protein in naïve NK cells from IRE1NK and IRE1f/f littermate control mice. (e) Quantitative real time PCR analysis of canonical c-Myc target genes in transferred Ly49H+ IRE1NK versus WT NK cells that were sorting-purified from the spleen of recipient Ly49H-deficient mice at day 1.5 PI. β-Actin was used as reference, and data are shown as the relative expression normalized to transferred WT NK cells. (f) Schematic of the putative XBP1 binding site in the c-Myc promoter region (based on SABiosciences’ proprietary database ENCODE). (g) Flow cytometric analysis of the kinetics of XBP1s, p-S6, p-Akt and pan-Akt expression in primary human NK cells after stimulation with IL-12 (20 ng/ml) and IL-18 (10 ng/ml) for the indicated time. (h) Purified splenic NK cells from IRE1NK and IRE1f/f littermate control mice were stimulated with mouse recombinant IL-12 (20 ng/ml) and IL-18 (10 ng/ml) for 1 hr and 16 hrs in the presence or absence of mTOR inhibitor rapamycin (10 nM). c-Myc levels were quantified by flow cytometry. (i) Flow cytometric analysis of c-Myc expression in transferred CD45.1 congenic WT and IRE1NK cells in the spleen of recipient Rag2−/−Il2rg−/− mice at specified time points after transfer. ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. One sample t-test performed on e. Two-way analysis of variance (ANOVA) with the Sidak post-test performed on h. Two tailed unpaired Student’s t-test performed on i. All error bars, mean with s.d. from biological replicates. n = 3~5 mice/group for all experiments and data were independently repeated two (c, g, h) or three times (d, e, i).
Supplementary Figure 7 Characterization of NK cell development and maturation in Myc+/− heterozygous mice (Het, referred to as ‘MycNK’ in Main Text) and Myc−/− (KO) mice.
(a) Quantitative real time PCR validation of c-Myc expression in Het and KO NK cells before and after IL-12 and IL-18 stimulation ex vivo. (b) Flow cytometric analysis of NK cell percentages and absolute numbers in BM and spleen of MycHet mice, in comparison to their Cre- littermate controls. BM NK cells are identified by Lin−CD122 and splenic NK cells are identified by Lin−NK1.1+. (c) as in b, except MycKO is shown. (d) Flow cytometric analysis of NK cell development in BM and NK cell maturation in spleen of MycHet mice. For BM (upper), percentages of NK cells that are NK progenitors (NKP, DX5− NK1.1−), immature NK cells (iNK, DX5− NK1.1+) or mature NK (mNK, DX5− NK1.1+) are shown; for spleen (bottom), percentages of NK cells that are immature (CD27+ CD11b−), mature (CD27− CD11b+) or intermediate stage (CD27+ CD11b+) are shown. (e) as in d, except MycKO is shown. (e) Representative histogram plots showing surface expression of cytokine receptors and activating receptors in splenic NK cells from MycHet mice in comparison to their Cre- littermate controls. (f) as in e, except MycKO is shown. p values as indicated. Two tailed unpaired Student’s t-test performed on a-e. All error bars, mean with s.e.m. Data are representative of three (a-e) and two (f, g) independent experiments. n = 3 mice/group for a, f, g. n = 4-5 mice/group for all experiments in b-e. Data were independently repeated three (b-e) or two (f, g) times.
Supplementary Figure 8 IRE1α-driven NK cell expansion is associated with the presence of immune cell types beneficial to tumor control.
(a) Representative flow cytometric plots and quantification of basal levels of NK cell numbers and relative percentage and Ki-67 and c-Myc expression in the lungs of naïve IRE1NK and IRE1f/f littermate control mice. (b) Gating strategy for c. (c) Percentage of conventional type 1 dendritic cells (cDC1) (left), CD8+ T cells (middle), and CD4+ T cells (right) in the lung and spleen of IRE1NK and IRE1f/f littermate control mice at day 20 following intravenous injection of B16F10 melanoma cells. * p < 0.05, ** p < 0.01. Two tailed unpaired Student’s t-test performed on c. All error bars, mean with s.e.m. n = 4 mice/group for a and 5 mice/group for c. Data were independently repeated three times.
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Dong, H., Adams, N.M., Xu, Y. et al. The IRE1 endoplasmic reticulum stress sensor activates natural killer cell immunity in part by regulating c-Myc. Nat Immunol 20, 865–878 (2019). https://doi.org/10.1038/s41590-019-0388-z
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