An XBP1s-PIM-2 positive feedback loop controls IL-15-mediated survival of natural killer cells

Abstract

Spliced X-box-binding protein 1 (XBP1s) is an essential transcription factor downstream of interleukin-15 (IL-15) and AKT signaling, which controls cell survival and effector functions of human natural killer (NK) cells. However, the precise mechanisms, especially the downstream targets of XBP1s, remain unknown. In this study, by using XBP1 conditional knockout mice, we found that XBP1s is critical for IL-15-mediated NK cell survival but not proliferation in vitro and in vivo. Mechanistically, XBP1s regulates homeostatic NK cell survival by targeting PIM-2, a critical anti-apoptotic gene, which in turn stabilizes XBP1s protein by phosphorylating it at Thr⁵⁸. In addition, XBP1s enhances the effector functions and antitumor immunity of NK cells by recruiting T-bet to the promoter region of Ifng. Collectively, our findings identify a previously unknown mechanism by which IL-15-XBP1s signaling regulates the survival and effector functions of NK cells.

Fig. 1.. XBP1s contributes to NK cell homeostasis in mice under ER stress.

(A) Representative plots (left) and summary data (right) of the percentages and absolute numbers of NK cells in the spleens from Xbp1^f/f and Xbp1^cKO mice treated with IL-15 for 5 days (n = 6 per group). (B) Representative plots (left) and summary data (right) of the percentages and the absolute numbers of NK cells in the spleens from Il15^TgXbp1^f/f and Il15^TgXbp1^cKO mice (n = 8 per group). (C) Representative plots (left) and summary data (right) of the percentages and the absolute numbers of NK cells in the lungs from B16F10 tumor-bearing Xbp1^f/f and Xbp1^cKO mice (n = 6 per group). (D) Representative plots (left) and summary data (right) of the percentages and the absolute numbers of NK cells in the brains from Xbp1^f/f and Xbp1^cKO mice at the onset of the EAE (n = 5 per group). Data are shown as means ± SD and were analyzed with an unpaired two-tailed t test. Data are representative of at least two independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 2.. XBP1s controls IL-15–mediated NK cell survival in vitro and in vivo.

(A) Percentages of annexin V⁺ NK cells in the spleens from Xbp1^f/f and Xbp1^cKO mice treated with IL-15 (n = 6 per group). (B) Percentages of annexin V⁺ NK cells in the spleens from Il15^TgXbp1^f/f and Il15^TgXbp1^cKO mice (n = 8 per group). (C) Splenic NK cells isolated from Il15^TgXbp1^f/f and Il15^Tg Xbp1^cKO mice were cultured in vitro in the presence of IL-15 (50 ng/ml), followed by cell counting with a trypan blue exclusion assay. (D) Live NK cells were counted at 4, 8, 12, and 24 hours after IL-15 withdrawal. Data shown are the fold change from the data collected at 0 hours. (E) Representative plots (left) and summary data (right) of apoptotic (annexin V⁺SYTOX Blue^+/−) NK cells from Xbp1^f/f and Xbp1^cKO mice 5 days after in vitro culture with IL-15 (50 ng/ml). (F) Representative plots (left) and summary data (right) of apoptotic (active caspase-3⁺) NK cells from Xbp1^f/f and Xbp1^cKO mice 5 days after in vitro culture with IL-15 (50 ng/ml). (G) Representative plots (left) and summary data (right) of apoptotic (FAM-FLICA⁺) NK cells from Xbp1^f/f and Xbp1^cKO mice 5 days after in vitro culture with IL-15 (50 ng/ml). Data are shown as means ± SD and were analyzed with an unpaired two-tailed t test (A, B, and E to G) or two-way ANOVA with the Holm-Šídák post-test (C and D). Data are representative of at least two independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 3.. XBP1s maintains NK cell survival by targeting PIM-2.

(A) Top 10 Gene Ontology clusters of DEGs in RNA-seq data. (B) Heat maps of top 20 genes expressed differentially between Il15^TgXbp1^f/f and Il15^TgXbp1^cKO mice. (C and D) qPCR (C) and immunoblotting (D) showing expression levels of PIM-2 in NK cells from Il15^TgXbp1^f/f and Il15^TgXbp1^cKO mice (n = 4 per group). (E) Integrative Genomics Viewer tracks displaying the distribution of XBP1s-binding peaks across the Pim2 transcript. (F and G) Luciferase reporter assay showing that XBP1s activates Pim2 gene transcription in HEK-293T cells (F) and mouse NK cells (G). (H and I) Binding of XBP1s to the Pim2 promoter in IL-15–treated Xbp1^f/f NK cells (H) or IL-15–treated Xbp1^cKO NK cells (I) was determined by ChIP-qPCR. (J) Scheme for retroviral transduction and BM chimeras. BM cells from Il15^TgXbp1^f/f and Il15^TgXbp1^cKO mice were infected with retrovirus encoding Pim2 or empty vector (EV). The infected cells were transferred into lethally irradiated CD45.1 recipient mice. After 8 weeks, mice were intraperitoneally injected with 2 μg of recombinant human IL-15 for 5 days. NK cells were isolated from the spleens of the chimeric mice and subjected to flow cytometry. (K) Summary data (left) and representative plots (right) of NK cells in chimeric mice (n = 3 per group). Data are shown as means ± SD and were analyzed with an unpaired two-tailed t test (C and F to I) or two-way ANOVA with the Holm-Šídák post-test (K). Data are representative of at least two independent experiments. ns, not significant; *P < 0.05; ***P < 0.001; ****P < 0.0001.

Fig. 4.. XBP1s maintains cell survival by targeting PIM-2 in human NK cells.

(A to C) qPCR (A) and immunoblotting (B and C) showing PIM-2 levels in human NK cells transduced with XBP1s lentivirus (n = 3 per group). (D) Scheme denoting putative XBP1s-binding sites in the PIM2 promoter. (E and F) Luciferase reporter assay showing that XBP1s activates PIM2 gene transcription in HEK-293T cells (E) and human NK cells (F). (G) Binding of XBP1s to the PIM2 promoter in human NK cells was determined by ChIP-qPCR. (H and I) Scheme for lentiviral transduction of PIM-2 into human NK cells and treatment with an inhibitor of IRE1α 4μ8C (50 μM) and IL-15 (50 ng/ml) (H). Live NK cell numbers were counted at the indicated time using a trypan blue exclusion assay [(I), n = 3 per group]. Data are shown as means ± SD and were analyzed with an unpaired two-tailed t test (A, C, and E to G) or one-way ANOVA with the Holm-Šídák post-test (I). Data are representative of at least two independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 5.. PIM-2 stabilizes XBP1s protein through phosphorylation at Thr⁵⁸.

(A) FLAG-tagged XBP1s and different doses of HA-tagged PIM-2 were transiently transfected into HEK-293T cells. XBP1s levels were analyzed by immunoblotting. (B and C) HEK-293T cells were transiently transfected with FLAG-tagged XBP1s or together with HA-tagged PIM-2 and further treated with CHX for the indicated time periods. XBP1s levels were analyzed by immunoblotting. Representative blots (B) and quantification of immunoblotting signals of XBP1s to actin levels (C) are shown. (D) HEK-293T cells were transiently transfected with the FLAG-tagged XBP1s and Ubb plasmids with or without the HA-tagged PIM-2 plasmid. Ubiquitinylated XBP1s levels were analyzed by immunoblotting. (E) Immunoblotting for HA-tagged PIM-2 and FLAG-tagged XBP1s proteins after immunoprecipitation of FLAG from HEK-293T cells. (F) Immunoblotting for HA-tagged PIM-2 and FLAG-tagged XBP1s proteins after immunoprecipitation of HA from HEK-293T cells. (G) IL-2–expanded mouse NK cells were treated with IL-15 (50 ng/ml) for 24 hours. Cell lysates were immunoprecipitated with rabbit anti-XBP1s or IgG isotype control and immunoblotted with rabbit anti–PIM-2 or mouse anti-XBP1s. (H) Primary human NK cells were treated with IL-15 (50 ng/ml) for 24 hours. Cell lysates were immunoprecipitated with rabbit anti-XBP1s or IgG isotype control and immunoblotted with rabbit anti–PIM-2 or mouse anti-XBP1s. (I) Immunoblotting using a phospho-Thr/Ser–specific antibody to detect phosphorylated XBP1s levels after immunoprecipitation of FLAG from HEK-293T cells. (J and K) FLAG-tagged XBP1s and HA-tagged PIM-2 (WT or KD PIM-2, K61A) were transfected into HEK-293T cells. Immunoblotting using phospho-Thr–specific (J) or phospho-Ser–specific antibody (K) for detecting phosphorylated XBP1s levels after immunoprecipitation of FLAG from HEK-293T cells. (L) FLAG-tagged WT XBP1s or Thr⁵⁸ mutant (T58A) XBP1s and HA-tagged PIM-2 were transfected into HEK-293T cells. Immunoblotting using phospho-Thr–specific antibody to detect phosphorylated XBP1s levels after immunoprecipitation of FLAG from HEK-293T cells. (M) HEK-293T cells were transiently transfected with FLAG-tagged XBP1s or T58A mutant XBP1s together with HA-tagged PIM-2 and further treated with CHX for 1 hour. XBP1s levels were analyzed by immunoblotting. Data are shown as means ± SD and were analyzed with two-way ANOVA with the Holm-Šídák post-test (C). Data are representative of at least two independent experiments. *P < 0.05; ****P < 0.0001.

Fig. 6.. XBP1s deficiency impairs NK cell effector functions and antitumor immunity.

(A to C) Expression levels of granzyme B (A), perforin (B), and IFN-γ (C) in NK cells from Il15^TgXbp1^f/f and Il15^TgXbp1^cKO mice (n = 8 per group). (D) Poly(I:C)-activated NK cells were isolated from the spleen and cocultured with YAC-1 cells at a ratio of 5:1, 2.5:1, and 1.25:1 for 4 hours. Cytotoxicity of NK cells was evaluated by standard ⁵¹Cr release assays (n = 3 per group). (E) Il15^TgXbp1^f/f and Il15^TgXbp1^cKO mice were intravenously injected with B16F10 cells (2 × 10⁵). Fourteen days after injection, mice were euthanized for postmortem analysis. Quantification of total metastatic nodules in the lung and the gross morphology of individual lung lobes are shown (n = 5 per group). (F) Il15^TgXbp1^f/f and Il15^TgXbp1^cKO mice were intravenously injected with B16F10 cells (2 × 10⁵) together with intraperitoneal injection of anti-NK1.1 antibody or IgG antibody (200 μg per mouse) on days 0 and 7 after tumor inoculation. The data show quantification of total metastatic nodules in the lung on day 14 (n = 5 per group). Data are shown as means ± SD and were analyzed with an unpaired two-tailed t test (A to E) or two-way ANOVA with the Holm-Šídák post-test (F). Data are representative of at least two independent experiments. **P < 0.01; ***P < 0.001.

Fig. 7.. XBP1s promotes IFN-γ production by recruiting T-bet to the promoter region of Ifng.

(A to C) qPCR analysis of mRNA levels of Ifng (A), Gzmb (B), and Prf1 (C) in NK cells isolated from tumor tissues of Il15^TgXbp1^f/f and Il15^TgXbp1^cKO tumor-bearing mice. (D) Luciferase reporter assay showing transcriptional activity of Gzmb after XBP1s overexpression. (E) Mouse XBP1s and T-bet were transfected alone or together into HEK-293T cells. The transcriptional activity of Ifng was examined by luciferase reporter assays. (F) Human XBP1s and T-BET were transfected alone or together into HEK-293T cells. The transcriptional activity of IFNG was examined by luciferase reporter assays. (G) Mouse XBP1s and T-bet were transfected alone or together into mouse NK cells. The transcriptional activity of Ifng was examined by luciferase reporter assays. (H) Human XBP1s and T-BET were transfected alone or together into human NK cells. The transcriptional activity of IFNG was examined by luciferase reporter assays. (I) IL-2–expanded mouse NK cells were treated with IL-15 (50 ng/ml) for 24 hours. Cell lysates were immunoprecipitated with rabbit anti–T-bet or IgG isotype control and immunoblotted with mouse anti-XBP1s or rabbit anti–T-bet. (J) Primary human NK cells were treated with IL-15 (50 ng/ml) for 24 hours. Cell lysates were immunoprecipitated with rabbit anti–T-bet or IgG isotype control and immunoblotted with mouse anti-XBP1s or rabbit anti–T-bet. (K) The cross-linked DNA-protein complexes in IL-15–activated mouse NK cells were separately immunoprecipitated using antibodies against XBP1s or T-bet. The binding of XBP1/T-bet complex to the Ifng promoter was determined by ChIP-qPCR. (L) The cross-linked DNA-protein complexes in IL-15–activated human NK cells were separately immunoprecipitated using antibodies against XBP1s or T-BET. The binding of XBP1/T-BET complex to the IFNG promoter was determined by ChIP-qPCR. (M and N) Scheme for lentiviral transduction of T-bet into mouse NK cells (M). Data shown are summary data (left) and representative plots (right) that came from quantifying IFN-γ production by transduced NK cells [(N), n = 4 per group]. Data are shown as means ± SD and were analyzed with an unpaired two-tailed t test (A to D) or one-way ANOVA with the Holm-Šídák post-test (E to H, K, and L) or two-way ANOVA with the Holm-Šídák post-test (N). Data are representative of at least two independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****p < 0.0001.