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. 2017 May 1;214(5):1493-1507.
doi: 10.1084/jem.20161524. Epub 2017 Mar 29.

The Kinase TBK1 Functions in Dendritic Cells to Regulate T Cell Homeostasis, Autoimmunity, and Antitumor Immunity

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Free PMC article

The Kinase TBK1 Functions in Dendritic Cells to Regulate T Cell Homeostasis, Autoimmunity, and Antitumor Immunity

Yichuan Xiao et al. J Exp Med. .
Free PMC article

Abstract

Dendritic cells (DCs) are crucial for mediating immune responses but, when deregulated, also contribute to immunological disorders, such as autoimmunity. The molecular mechanism underlying the function of DCs is incompletely understood. In this study, we have identified TANK-binding kinase 1 (TBK1), a master innate immune kinase, as an important regulator of DC function. DC-specific deletion of Tbk1 causes T cell activation and autoimmune symptoms and also enhances antitumor immunity in animal models of cancer immunotherapy. The TBK1-deficient DCs have up-regulated expression of co-stimulatory molecules and increased T cell-priming activity. We further demonstrate that TBK1 negatively regulates the induction of a subset of genes by type I interferon receptor (IFNAR). Deletion of IFNAR1 could largely prevent aberrant T cell activation and autoimmunity in DC-conditional Tbk1 knockout mice. These findings identify a DC-specific function of TBK1 in the maintenance of immune homeostasis and tolerance.

Figures

Figure 1.
Figure 1.
DC-specific TBK1 ablation has no effect on DC development. (A) Immunoblot analysis of phosphorylated TBK1 (p-TBK1) and total TBK1 in whole-cell lysates of BMDCs stimulated with 100 ng/ml LPS (left) and splenic DCs (Spl DC) from young (Y; 6 wk old) or old (O; 16 wk old) WT mice (right). (B) PCR analysis of TBK1 and CD11c-Cre to identify the genotype of WT, heterozygous (Het), and homozygous DC-conditional Tbk1 KO (DKO) mice (left) and immunoblot analysis of TBK1 protein expression in BMDCs from WT and Tbk1-DKO mice (right). (C) Flow cytometric analysis of the frequency (numbers in quadrants) of total DCs (CD11c+), plasmacytoid DCs (pDC; CD11c+PDCA1+), macrophages (MΦ; F4/80+CD11b+), and neutrophils (NP; Gr-1+CD11b+) in the bone marrow of WT and Tbk1-DKO mice. SSC, side scatter. (D–G) Flow cytometric analysis of the frequency of conventional DC (cDC; CD11c+MHCII+; D), plasmacytoid DC (CD11c+PDCA1+; E), CD8α+ DC (CD11c+CD8α+; F), and migratory (M-DC; MHCIIhiCD11c+) and resident (R-DC; MHCIIintCD11c+) DC (G) populations in the spleen (D–F) or cutaneous LNs (G) of WT and Tbk1-DKO mice, presented as representative plots (top) and summary graphs (bottom). Data are representative of at least three independent experiments.
Figure 2.
Figure 2.
TBK1 deficiency in DCs impairs T cell homeostasis and causes autoimmunity. (A) Flow cytometric analysis of thymocytes in Tbk1-DKO and WT control mice, with the percentage of double-negative (DN), double-positive (DP), and CD4+ and CD8+ single-positive (SP) populations summarized based on three WT and three Tbk1-DKO mice (8 wk old). Data are presented as means ± SD. (B and C) Flow cytometric analysis of the frequency and absolute number of naive (CD44loCD62Lhi) and memory-like (CD44hiCD62Llo for CD4+ and CD44hi for CD8+ T cells) CD4+ and CD8+ T cells in total splenocytes from WT and Tbk1-DKO mice (10 wk and 8 mo old). Data are presented as representative plots (left) and summary graphs (right). (D) Flow cytometric analysis of the percentage of IFN-γ–producing and IL-17–producing CD4+ and CD8+ T cells in the spleen of 8-mo-old WT and Tbk1-DKO mice. (E) Frequency of CD4+Foxp3+ T reg cells in the thymus (Thy), spleen (Spl), and inguinal lymph nodes (ILN) of 10-wk-old WT and Tbk1-DKO mice, presented as a representative FACS plot (left) and summary graph based on multiple mice (right). (F and G) Representative images (F) and total cell number (G) of spleen, peripheral lymph nodes (pLN), and mesentery lymph nodes (mLN) of WT and Tbk1-DKO mice (8 mo old). (H) Hematoxylin-eosin staining of the indicated tissue sections from 8-mo-old WT and Tbk1-DKO mice, showing immune cell infiltrations in the Tbk1-DKO tissues (arrows). Bars, 100 µm. Data are representative of three or more independent experiments. *, P < 0.05; **, P < 0.01.
Figure 3.
Figure 3.
DC-specific TBK1 deletion promotes T cell responses in EAE induction. (A) Mean clinical scores of age- and sex-matched WT and Tbk1-DKO mice (8 wk old) subjected to MOG35–55-induced EAE. n = 5/group. (B and C) Flow cytometric analysis of T cells infiltrating into the CNS (brain and spinal cord) of day-15 EAE-induced mice, presented as representative plots (B) and absolute number (C). (D and E) Frequency and absolute number of IFN-γ– and IL-17–producing effector T cells in the CNS of day-15 MOG35–55-immunized WT and Tbk1-DKO mice, shown as a representative plot (D) and a summary graph (E). (F and G) Frequency and absolute number of CD62LloCD44hi and CD69+ CD4+ effector T cells in the spleen of day-15 MOG35–55-immunized mice, presented as representative plots (F) and summary graphs (G). (H) Frequency of spleen T reg cells from day-15 EAE–induced mice. (I and J) Frequency and absolute number of IFN-γ– and IL-17–producing CD4+ T cells in the draining lymph node (dLN) of day-15 EAE-induced mice. (K) Thymidine incorporation cell proliferation assays of splenic T cells from day-15 EAE-induced mice, stimulated in vitro with the indicated concentrations of MOG peptide. Data are representative of at least three independent experiments and are presented as means ± SD. *, P < 0.05; **, P < 0.01.
Figure 4.
Figure 4.
TBK1 deficiency in DCs promotes antitumor immunity. (A and B) Tumor growth (A) and survival (B) curves of WT and Tbk1 DKO mice injected s.c. with B16-OVA melanoma cells. n = 10. (C and D) Flow cytometric analysis of the frequency (C) and absolute cell numbers (D) of IFN-γ–producing CD4+ and CD8+ T cells in tumors of WT and Tbk1-DKO mice injected s.c. with B16-OVA melanoma cells (day 16 after injection). (E and F) Flow cytometric analysis of the percentage and absolute cell numbers of IFN-γ–producing CD4+ and CD8+ T cells (E) or percentage of T reg cells (F) in draining lymph nodes of WT and Tbk1-DKO mice injected s.c. with B16-OVA melanoma cells (day 16 after injection). Cells were stimulated with OVA peptide for 5 h after intracellular staining. (G–J) Tumor growth (G and I) and flow cytometric analysis of IFN-γ–producing CD4+ and CD8+ T cells in draining lymph nodes (H and J) of WT and Tbk1-DKO mice injected s.c. with EG7-OVA (G and H) or EL4 (I and J) thymoma cells (day 22 for EG7-OVA and day 16 for EL4 after injection). (K and L) Tumor growth (K) and survival (L) curves of WT and Tbk1-DKO mice injected s.c. with B16 melanoma cells without the surrogate antigen OVA (n = 10) followed by i.p. injection with PD-1 antibody on days 7, 10, and 13. Ctrl, control. (M) Tumor growth curve of WT mice injected s.c. with B16-OVA melanoma cells and then treated (on day 7) i.v. with WT or Tbk1-DKO BMDCs that were pulsed with OVA peptide and matured with LPS. (N) Flow cytometric analysis of the absolute cell numbers of IFN-γ–producing CD4+ and CD8+ T cells in tumors of the mice from M (day 18 after injection). (O and P) CFSE-labeled WT BMDCs and efluor450-labeled Tbk1-DKO BMDCs were subjected to flow cytometry analysis (O) or injected into day-7 B16 tumor-bearing WT mice for 18 h followed by flow cytometric detection of the transferred DCs in spleen, draining LNs (dLN), or tumor (P). Data are representative of at least three independent experiments and are presented as means ± SD. *, P < 0.05; **, P < 0.01.
Figure 5.
Figure 5.
TBK1 regulates the expression of IFN-response genes in DCs. (A) Flow cytometric analysis of phosphorylated TBK1 (P-TBK1) in CD11c+ DCs isolated from the draining LNs (dLN) of untreated (naive) mice or mice injected s.c. with B16 melanoma cells. (B and C) Flow cytometric analysis of CD80 and CD86 expression on WT or Tbk1-DKO spleen CD11c+ DCs (Spl DC) and B16 tumor-infiltrating CD11c+ DCs (Tumor DC). MFI, mean fluorescence intensity. (D) Flow cytometric analysis to measure the proliferation of CFSE-labeled OTII T cells incubated with either medium control or OVA-pulsed WT and Tbk1-DKO spleen DCs. (E) qRT-PCR analysis of the indicated genes using LPS-stimulated splenic DCs derived from WT and Tbk1-DKO mice (8 wk old). (F–I) RNA-sequencing analysis using splenic DCs freshly isolated from 8-wk-old WT and Tbk1-DKO mice (each group has three samples), showing a heat map of highly variable genes (top 1,500; F), genes with adjusted p-value <0.01 and log2 fold-change >1.5 (G), IFN-responsive genes (H), and NF-κB signature genes using the IFN-responsive gene Ifi202b as a positive control (I). (J) qRT-PCR analysis of the indicated genes in freshly isolated splenic DCs from WT and Tbk1-DKO mice (8 wk old). (K) qRT-PCR analysis of the indicated genes using splenic DCs from J that were starved for 6 h and then either not treated (NT) or stimulated with IFN-β for 3 h. Data are representative of three independent experiments and are presented as means ± SD. *, P < 0.05; **, P < 0.01.
Figure 6.
Figure 6.
Deletion of IFNAR1 prevents autoimmunity and suppresses antitumor immunity in Tbk1 DKO mice. (A) Frequency of naive (CD44loCD62Lhi) and memory (CD44hiCD62Llo) CD4+ T cells and IFN-γ–producing CD4+ T cells in the spleen of age- and sex-matched mice with the indicated genotype (4 mo old). (B) Summary data of flow cytometric analysis of memory CD4+ (CD4+CD44hiCD62lo) and CD8+ (CD8+CD44hi) T cells in the spleen described in A. (C) Representative spleen and peripheral lymph node (pLN) images of age- and sex-matched mice with the indicated genotype (4 mo old). (D) Hematoxylin-eosin staining of the indicated tissue sections from age- and sex-matched mice (5 mo old), showing immune cell infiltrations in the Tbk1-DKO tissues (arrows). Bars, 100 µm. (E) Growth curve of tumors (n = 5) of WT mice that were injected s.c. with B16-OVA melanoma cells and then treated i.v. by BMDCs with the indicated genotype that were pulsed with OVA peptide and matured with LPS. Data are representative of at least three independent experiments and are presented as means ± SD. *, P < 0.05; **, P < 0.01.
Figure 7.
Figure 7.
TBK1 mediates STAT3 phosphorylation in DCs. (A and B) Flow cytometric analysis of tyrosine (Y701) and serine (S727) phosphorylation of STAT1 and STAT3 in freshly isolated splenic CD11c+ DCs from 8-wk-old Ifnar+/+ and Ifnar−/− mice (A) or 8-wk-old WT and Tbk1-DKO mice (B). (C) qRT-PCR analysis of the indicated genes in freshly isolated splenic CD11c+ DCs from age- and sex-matched WT and Stat3-DKO mice (8 wk old). (D) Flow cytometric analysis of naive (CD44loCD62Lhi) and memory-like (CD44hiCD62Llo) CD4+ and CD8+ T cells in splenocytes from age- and sex-matched WT and Stat3-DKO mice (3 mo old). (E) Sequence alignment of the S727 phosphorylation site of STAT3 with the phosphorylation site of several known TBK1 substrate proteins. Conserved residues are shown in red, and the phosphorylation serine is underlined. MAVS, mitochondrial antiviral-signaling protein. (F) Lysates of WT or Tbk1-DKO splenic DCs were subjected to IP using anti-TBK1 or a control Ig (Ctrl); TBK1 and TBK1-associated STAT3 were detected by immunoblotting (IB). (G) Immunoblot analysis of STAT3 phosphorylation in HEK293T cells transfected with STAT3 along with either TBK1 or a catalytically inactive TBK1 mutant (TBK1M). (H) Immunoblot analysis of S727 phosphorylated STAT3, total STAT3, and loading control HSP60 in BMDCs prepared from STAT3-WT, Stat3-DKO, or STAT3-S727A mutant (STAT3-SA) mice, either not treated (NT) or stimulated with LPS. (I) Tumor growth curve of WT mice injected s.c. with B16-OVA melanoma cells and then injected i.v. (on day 7) with BMDCs described in H that were pulsed with OVA peptide and matured with LPS. (J and K) Tumor-infiltrating CD4+ (J) and CD8+ (K) T cells from the tumor-bearing mice described in I, presented as representative FACS plots (left) and summary graphs based on multiple mice (right). Data are representative of three or more independent experiments and are presented as means ± SD. *, P < 0.05; **, P < 0.01.

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