PMC

Tbkbp1 recruits PP6 to Ulk1 to oppose mTORC1-mediated Ulk1 phosphorylation. a Co-IP analysis of mTOR-Ulk1 interactions (upper panels) and direct IB analysis of the indicated proteins (lower panels) in NKT hybridoma cells transduced with a control or Tbkbp1-specific shRNA, either not treated (NT) or incubated with IL-15 or the mTORC1 inhibitor rapamycin (Rap). IgG was used as a negative control for IP. b Co-IP analysis of Tbkbp1-ANKRD28 interactions and direct IB assays using lysates of HEK293 cells transfected with the indicated expression vectors. c Co-IP analysis of ULK1 association with Tbkbp1 and ANKRD28 using lysates of HEK293 cells transfected with the indicated expression vectors. d Co-IP analysis of Ulk1 interaction with FIP200, Tbkbp1, and ANKRD28 using lysates of IL-15-stimulated NKT hybridoma cells transduced with a control or Tbkbp1-specific shRNA. IgG was used as a negative control for IP. e, f IB analysis of the indicated phosphorylated (P-) and total proteins in whole-cell lysates of IL-15-stimulated NKT hybridoma cells transduced with a nonsilencing control shRNA, two different ANKRD28-specific shRNAs (e) or two different PP6c-specific shRNAs (f). Data are representative of three independent experiments

Tbkbp1 facilitates AMPKa-mediated Ulk1 activation by antagonizing mTORC1. a, b IB analysis of the indicated phosphorylated (P-) and total proteins in whole-cell lysates of IL-15-stimulated WT and Tbkbp1-KO thymic NKT cells (a) or NKT hybridoma cells transduced with a non-silencing control shRNA or two different Tbkbp1-specific shRNAs (b). c IB analysis of the indicated phosphorylated (P-) and total proteins in whole-cell lysates of IL-15-stimulated NKT hybridoma cells. d, e IB analysis of phosphorylated Ulk1 on Ser757 or Ser555 using WT or Tbkbp1-KO NKT cells incubated for 60 min with the mTOR inhibitor Torin or solvent control DMSO (d) or IL-15-stimulated NKT hybridoma cells transduced with a nonsilencing control shRNA or two different Tbkbp1-specific shRNAs (e). f IB analysis of phosphorylated Ulk1 on Ser757 or Ser 555 in IL-15-stimulated NKT hybridoma cells transduced with an empty vector or HA-Tbkbp1 expression vector. g Co-IP analyses of AMPKα-Ulk1 interactions (upper panels) and direct IB analyses of the indicated proteins (lower panels) in NKT hybridoma cells transduced with a control or Tbkbp1-specific shRNA (#1) incubated with IL-15 as indicated. IgG was used as a negative control for IP. Data are representative of three or more independent experiments

Tbkbp1 mediates IL-15-stimulated NKT cell survival. a, b Flow cytometric analysis of NKT cell frequency in NKT-enriched WT and Tbkbp1-KO thymocytes (CD8⁺ cells depleted by microbeads) after in vitro incubation with IL-15 for the indicated time periods. Data are presented as a representative plot (a) and summary graph (b). c, d Flow cytometry analysis of apoptotic cells (based on AnnexinV and PI staining) in enriched thymic NKT cells cultured for indicated time periods with IL-15 or medium control. Data are presented as a representative plot (c) and summary graph (d). e Proliferation assays (based on CFSE dilution) of WT or Tbkbp1-KO thymic NKT cells labeled with CFSE and cultured for the indicated time with IL-15. f, g Flow cytometric analysis of intracellular Bcl-2 level (f) and qRT-PCR analysis of Bcl2 mRNA (g) in WT or Tbkbp1-KO thymic NKT cells, either freshly isolated or cultured with IL-15 for 48 h, presented as a representative plot (left) and summary graph (right). All summary graphs are presented as mean ± s.d. values based on 5 WT and 5 Tbkbp1-KO mice. The similar data were obtained from at least three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. Mann–Whitney test (b), Student’s t-test (d, g)

Tbkbp1-mediated NKT cell survival involves regulation of mitochondria ROS. a IB analyses of the indicated phosphorylated (P-) and total proteins of NKT cells stimulated with IL-15 for the indicated time periods. b Flow cytometric analysis of WT and Tbkbp1-KO thymic NKT cells that were stained with CellROX^TM Deep Red and MitoSOX for detection of ROS or stained with MitoTracker Green and MitoTracker Red CMXRos for detection of mitochondrial mass and membrane potential, respectively. c, d Flow cytometric analysis of ROS concentration in WT and Tbkbp1-KO thymic NKT cells that either untreated (0 h) or cultured for 2 days with IL-15 or IL-15 plus NAM. Data are presented as a representative plot (c) and summary graph (d). n = 5 per genotype group. e, f Flow cytometric analysis of intracellular Bcl-2 or Bcl-xL level in WT and Tbkbp1-KO thymic NKT cells that either untreated (0 h) or cultured for 2 days with IL-15 or IL-15 plus NAM. Data are presented as representative plots (e) and summary graphs (f). n = 5 mice per genotype group. g Flow cytometry quantification of AnnexinV⁺ apoptotic cells in thymic NKT cells cultured for 2 days with medium control, IL-15, or IL-15 plus NAM, presented as a summary graph. n = 5 mice per genotype group. Data are representative of three independent experiments, and bar graphs are presented as mean ± s.d. values. *P < 0.05; **P < 0.01. Student’s t-test (d, f, g)

Tbkbp1 deficiency increases the frequency of memory-like CD8⁺ T cells and antitumor immunity. a, b Flow cytometric analysis of memory-like CD8⁺ T cells based on expression of CD44, CD122, and CXCR3 surface markers in the CD8⁺ single positive (SP) thymocytes (a) and spleen CD8⁺ T cells (b) derived from 6-week-old WT and Tbkbp1-KO (KO) mice, presented as representative FACS plots (upper) and summary graphs (lower, each circle represents a mouse). c, d ICS analysis of Emoes⁺ and T-bet⁺ cells (c) and IFNγ⁺ and IFNγ⁺Eomes⁺ cells (d) in CD8⁺ single positive (SP) thymocytes. e, f Tumor growth (e) and survival (f) curves of WT and Tbkbp1-KO (KO) mice injected s.c. with B16-OVA melanoma cells. Lethality was defined as tumor size reaching to 225 mm². n = 8 per genotype group. g–j Flow cytometric analysis of the frequency of IFNγ-producing CD8⁺ (g, i) and CD4⁺ (h, j) T cells in the tumors (g, h) or draining lymph node (i, j) of WT and Tbkbp1-KO mice injected s.c. with B16-OVA melanoma cells (day 15 after injection), presented as representative plots (left) and summary graphs (right). Data are representative of at least three independent experiments, and bar graphs are presented as mean ± s.d. values. *P < 0.05; **P < 0.01; ***P < 0.001. Student’s t-test (b, g, i), Mann–Whitney test (a, h, j), Two-way ANOVA (e), log-rank Mantel-Cox test (f)

Tbkbp1 has a cell-intrinsic role in regulating NKT cell development. a, b Flow cytometric analysis of NKT cells in the thymus, spleen, and liver from 6-week-old WT and Tbkbp1-TKO (TKO) mice, presented as a representative FACS plot of thymic NKT cells (a) and summary graphs of the indicated NKT cells (each circle represents a mouse) (b). c, d Flow cytometric analysis of thymic NKT cell maturation stages (stage 1: CD44^–NK1.1^–; stage 2: CD44⁺NK1.1^–; stage 3: CD44⁺NK1.1⁺), presented as a representative plot (c) and summary graphs (d). e Flow cytometric analysis of IL-4 expression in WT and Tbkbp1-TKO thymic NKT cells after treatment for 4 h with PMA and ionomycin in the presence of monensin, presented as a representative plot and summary graph (n = 5 per genotype). f, g Flow cytometric analysis of NKT cells and their maturation stages in the thymus (f) and spleen (g) of Rag1-KO recipient mice adoptively transferred (for 6 week) with a mixture of BM cells derived from WT B6.SJL mice (CD45.1⁺) and Tbkbp1-KO mice (CD45.2⁺), gating on CD45.1⁺ or CD45.2⁺ cells and presented as representative FACS plots and summary graphs (n = 5 chimeric mice). h Flow cytometric analysis of cell surface expression of CD1d in DP thymocytes from WT or Tbkbp1-KO mice, presented as a representative FACS plot and summary graph (n = 5 mice per genotype). i ELISA of IL-2 produced by NKT hybridoma cells cocultured for 24 h with total thymocytes from WT or Tbkbp1-KO mice in the absence or presence of α-GalCer. Data are representative of at least three independent experiments, and bar graphs are presented as mean ± s.d. values. *P < 0.05; **P < 0.01; ***P < 0.001. Student’s t-test (b, d–i)

Tbkbp1 is abundantly expressed in NKT cells and has a cell-intrinsic role in regulating NKT cell development. a qRT-PCR analyses of Tbkbp1 mRNA in total, CD4⁺CD8⁺ double-positive (DP), CD4^–CD8^– double-negative (DN), and NKT thymic populations (upper) or splenic CD4⁺ and CD8⁺ T cells, NKT cells, and NK cells (lower). b IB analysis of Tbkbp1 and loading control Tubulin in the indicated splenic cell populations. c Flow cytometric analysis of NKT cell frequency and absolute numbers in the thymus (Thy), spleen (Spl) and liver (Liv) of age-matched WT and Tbkbp1-KO (KO) mice, presented as representative plots (left) and summary graphs (right). n = 6 per genotype. d, e Flow cytometric analysis of NKT cell maturation stages (stage1: NK1.1^–CD44^–; stage2: NK1.1^–CD44⁺; stage 3: NK1.1⁺CD44⁺) in the thymus, spleen and liver of WT and Tbkbp1-KO mice, presented as representative plots (d) and summary graphs (e). n = 6 per genotype. f, g Flow cytometric analysis of the indicated transcription factors in thymic (f) and splenic (g) NKT cells from WT and Tbkbp1-KO mice, presented as representative plots (left) and summary graphs based on PLZF/RORγt flow values (right). n = 6 per genotype. h Flow cytometric analysis of thymic NKT cells based on IL-17Rb expression. i Flow cytometric analysis of IFNγ and IL-4 expression in WT and Tbkbp1-KO thymic NKT cells after treatment for 4 h with PMA and Ionomycin in the presence of monensin, presented as representative plots and summary graphs (n = 6 per genotype). j ELISA of IFN-γ and IL-4 in the supernatant of WT and Tbkbp1-KO thymic NKT cells after 48 h of in vitro stimulation with α-Galcer and antigen-presenting cells (WT BMDCs). k ELISA of IFNγ and IL-4 in the serum of WT and Tbkbp1-KO mice injected with α-Galcer (4 μg) for 6 h (n = 5 per genotype). Data are representative of three independent experiments, and bar graphs are presented as mean ± s.d. values. *P<0.05; **P<0.01; ***P<0.001. One-way ANOVA (a), Mann–Whitney test (e, i), or Student’s t-test (c, f, g, j, k)

Tbkbp1 mediates IL-15-stimulated autophagy in NKT cells. a–d IB analysis of LC3 modification and p62 degradation (a, c) and Ulk1 phosphorylation (b, d) in whole-cell lysates of IL-15-stimulated WT or Tbkbp1-KO thymic NKT cells (a, b) or NKT hybridoma cells transduced with a non-silencing control shRNA (shCtrl) or two different Tbkbp1-specific shRNAs that were stimulated with IL-15 (d) or IL-15 together with DMSO or bafilomycin A (c). e Confocal microscopic analysis of LC3 puncta, DAPI (nuclear staining), and merged picture of untreated (NT) or IL-15-stimulated control or Tbkbp1-knockdown NKT hybridoma cells. Data are presented as representative images (left) and summary graph of quantified LC3 puncta (right). Scale bar, 5 μm. f Flow cytometric analysis of intracellular Bcl2 expression level in WT or Tbkbp1-KO thymic NKT cells cultured for 2 days with medium control, IL-15, or IL-15 plus the autophagy inhibitor 3MA. g Summary graph of flow cytometric analysis of AnnexinV⁺ apoptotic cells in enriched thymic NKT cells that were either untreated (NT) or cultured for 1 day with the indicated agents. h, i Co-IP analysis of Tbkbp1-FIP200 (h) or Tbkbp1-Ulk1(i) interaction (upper) and direct IB assays (lower) using lysates of HEK293 cells transfected with the indicated expression vectors. j IB analysis of the indicated proteins in IP samples of control IgG, anti-FIP200, or anti-Ulk1 or direct lysates of NKT hybridoma cells that were stimulated as indicated. k Schematic of Tbkbp1 WT and mutants showing the coiled-coil domains (CCs), TBK-binding domain (TBD), proline-rich domain, and zinc fingers (ZF). l, m Co-IP analysis of Ulk1 binding with the indicated Tbkbp1 mutants (upper) and direct IB assays (lower) using HEK293 cells transfected with Myc-ULk1 along with Tbkbp1 truncation mutants (l) and point mutants (m). n IB analysis of the indicated phosphorylated (P-) or total proteins in whole-cell lysates of Tbkbp1-knockdown NKT hybridoma cells reconstituted with a vector control (Ctrl) or the indicated Tbkbp1 point mutants, stimulated as indicated. Data are representative of three or more independent experiments. **P < 0.01; ***P < 0.001. Two-way ANOVA (e), Student’s t-test (g)