Letter | Published:

Hippo/Mst signalling couples metabolic state and immune function of CD8α+ dendritic cells

Naturevolume 558pages141145 (2018) | Download Citation

Abstract

Dendritic cells orchestrate the crosstalk between innate and adaptive immunity. CD8α+ dendritic cells present antigens to CD8+ T cells and elicit cytotoxic T cell responses to viruses, bacteria and tumours1. Although lineage-specific transcriptional regulators of CD8α+ dendritic cell development have been identified2, the molecular pathways that selectively orchestrate CD8α+ dendritic cell function remain elusive. Moreover, metabolic reprogramming is important for dendritic cell development and activation3,4, but metabolic dependence and regulation of dendritic cell subsets are largely uncharacterized. Here we use a data-driven systems biology algorithm (NetBID) to identify a role of the Hippo pathway kinases Mst1 and Mst2 (Mst1/2) in selectively programming CD8α+ dendritic cell function and metabolism. Our NetBID analysis reveals a marked enrichment of the activities of Hippo pathway kinases in CD8α+ dendritic cells relative to CD8α dendritic cells. Dendritic cell-specific deletion of Mst1/2—but not Lats1 and Lats2 (Lats1/2) or Yap and Taz (Yap/Taz), which mediate canonical Hippo signalling—disrupts homeostasis and function of CD8+ T cells and anti-tumour immunity. Mst1/2-deficient CD8α+ dendritic cells are impaired in presentation of extracellular proteins and cognate peptides to prime CD8+ T cells, while CD8α dendritic cells that lack Mst1/2 have largely normal function. Mechanistically, compared to CD8α dendritic cells, CD8α+ dendritic cells exhibit much stronger oxidative metabolism and critically depend on Mst1/2 signalling to maintain bioenergetic activities and mitochondrial dynamics for their functional capacities. Further, selective expression of IL-12 by CD8α+ dendritic cells depends on Mst1/2 and the crosstalk with non-canonical NF-κB signalling. Our findings identify Mst1/2 as selective drivers of CD8α+ dendritic cell function by integrating metabolic activity and cytokine signalling, and highlight that the interplay between immune signalling and metabolic reprogramming underlies the unique functions of dendritic cell subsets.

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Acknowledgements

The authors acknowledge N. Chapman for critical reading of the manuscript, R. Johnson for Stk4 and Stk3 floxed mice, E. Olson for Yap1 and Taz floxed mice, S.C. Sun for the NIK(Δ78–84) construct, M. Hendren and A. KC for animal work, SJCRH Electron Microscopy core resource for electron microscopy, and Immunology FACS core facility for cell sorting. This work was supported by NIH AI105887, AI101407, CA176624, CA221290, NS064599 (to H.C.), and AG047928 (to J.P.).

Reviewer information

Nature thanks L. O’Neill and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Affiliations

  1. Department of Immunology, St Jude Children’s Research Hospital, Memphis, TN, USA

    • Xingrong Du
    • , Jing Wen
    • , Yanyan Wang
    • , Peer W. F. Karmaus
    • , Cliff Guy
    • , Thanh-Long M. Nguyen
    • , Yogesh Dhungana
    •  & Hongbo Chi
  2. Department of Computational Biology, St Jude Children’s Research Hospital, Memphis, TN, USA

    • Alireza Khatamian
    •  & Jiyang Yu
  3. Departments of Structural Biology and Developmental Neurobiology, St Jude Children’s Research Hospital, Memphis, TN, USA

    • Haiyan Tan
    • , Yuxin Li
    •  & Junmin Peng
  4. St Jude Proteomics Facility, St Jude Children’s Research Hospital, Memphis, TN, USA

    • Haiyan Tan
    • , Yuxin Li
    •  & Junmin Peng
  5. Hartwell Center for Bioinformatics and Biotechnology, St Jude Children’s Research Hospital, Memphis, TN, USA

    • Geoffrey Neale

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Contributions

X.D. designed and performed in vitro and in vivo experiments, and wrote the manuscript; J.W. performed human DC study and immunoprecipitation; Y.W. initiated the project, and together with P.W.F.K. performed Seahorse assays; A.K. performed network inference; H.T., Y.L. and J.P. performed and analysed proteomics assays; C.G. performed STORM assays and helped with electron microscopy experiments; T.-L.M.N. performed the tumour study; Y.D. and G.N. performed functional enrichment analysis of microarrays; J.Y. developed the NetBID algorithm, performed systems biology analysis, wrote the manuscript and supervised overall computational analysis; and H.C. designed experiments, wrote the manuscript, and provided overall direction.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Jiyang Yu or Hongbo Chi.

Extended data figures and tables

  1. Extended Data Fig. 1 NetBID analysis for the reconstruction of the DC signalling interactome (DCI), network and enrichment analyses of top kinase drivers, and identification and validation of Stk4 (Mst1) regulons.

    a, PCA plot of baseline microarray gene expression profiles of total DCs (blue, n = 15; used for de novo DCI reconstruction), CD8α+ and CD8α DCs (green and red, respectively, n = 4 each; used for differential expression analysis) after removal of batch effects. b, Top 36 hub kinases that are differentially activated in CD8α+ DCs relative to CD8α DCs inferred by NetBID. Left, the NetBID panel indicates the significance level (colour coded by z score; labelled values are P values) of the driver network in integrated analysis, transcriptomics (mRNA), whole proteomics (wProtein) and phosphoproteomics (pProtein) data, respectively. Right, differential expression of the drivers (colour coded by z score; labelled values are signed fold changes). The Venn diagram shows the enrichment of Hippo pathway kinases in the top putative kinase drivers. c, Network interactions of top 36 kinase drivers of CD8α+ DCs. d, Top signalling pathways enriched by 36 NetBID-inferred kinase drivers (P < 0.01, number of overlapped genes >2). e, Known kinases in Hippo signalling9 and analysis by NetBID. f, Stk4-mediated gene network (n = 140) from DCI computationally inferred from baseline gene expression profiles of total DCs by NetBID. The width of an edge is proportional to the pairwise mutual information of connected nodes. g, h, Enrichment of predicted Mst1 signalling regulons (as shown in f) in differentially expressed genes between Mst1/2-deficient (Mst1/2∆DC) and wild-type CD8α+ DCs (g) or CD8α DCs (h). Pval.GSEA indicates the P value of GSEA; Pval.Stk4 and FC.Stk4 indicate the P value and signed fold change of Stk4 expression (insert).

  2. Extended Data Fig. 2 T cell homeostasis in mice with DC-specific deletion of Hippo pathway genes.

    a, b, Total cellularity (a) or T cell numbers (TCRβ+ CD8+ and TCRβ+ CD4+) (b) of the spleen, peripheral lymph nodes (PLN) and mesenteric lymph nodes (MLN) of wild-type and Mst1/2∆DC mice (n = 5 mice per genotype). c, Flow cytometry analysis of splenic CD4+ and CD8+ T cell populations (upper) and frequencies of total CD4+ and CD8+ T cells in spleen, PLN and MLN (lower) of wild-type and Mst1/2∆DC mice (n = 8 mice per genotype). d, CD44 and CD62L expression on splenic CD4+ and CD8+ T cells of wild-type and Mst1/2∆DC mice. e, CD44 and IFNγ expression in splenic CD4+ and CD8+ T cells. fk, Flow cytometry analysis of CD4+ and CD8+ populations, expression of CD44 and CD62L, or CD44 and IFNγ in CD4+ and CD8+ T cells from spleen of wild-type and Lats1/2∆DC (fh) or Yap/Taz∆DC mice (ik). ln, Frequencies of CD44high CD62Llow effector/memory cells (l) and CD44+ IFNγ+ cells (m) in splenic CD4+ and CD8+ T cells and frequencies of splenic CD4+ and CD8+ T cells (n) of wild-type (n = 9), Cd11ccreStk4fl/flStk3+/+ (n = 5), Cd11ccreStk4+/+Stk3fl/fl (n = 4), Cd11ccreStk4fl/+Stk3fl/fl (n = 5) and Cd11ccreStk4fl/flStk3fl/+ (n = 4) mice (Cd11c is also known as Itgax). Numbers in quadrants or gates indicate percentage of cells. Data are mean and s.e.m. NS, not significant; *P < 0.05; **P < 0.01; two-tailed unpaired Student’s t-test in ac; one-way ANOVA in ln. Data summarize two (ik), three (fh), four (a, b, d, e), six (c) or eight (ln) independent experiments.Source Data.

  3. Extended Data Fig. 3 Analyses of Mst1 and Mst2 deletion in DCs and lymphocytes from Mst1/2∆DC mice and T cell homeostatic status in mixed bone marrow chimaeras.

    a, Real-time PCR (upper and middle) and immunoblot (lower) analyses of Stk4 (also known as Mst1) and Stk3 (also known as Mst2) mRNA and protein expression in CD4+ T cells, CD8+ T cells and B cells from wild-type and Mst1/2∆DC mice (n = 3 mice per genotype). b, Real-time PCR analysis of Stk4 and Stk3 mRNA expression in splenic CD8α+ and CD8α DCs from wild-type and Mst1/2∆DC mice (n = 3 for accessing Mst2 expression in Mst1/2-deficient CD8α+ DCs, n = 4 for others). c, d, Bone marrow cells from wild-type or Mst1/2∆DC CD45.2.2+ mice were mixed with cells from CD45.1.2+ (spike) mice at a 1:1 ratio and transferred into lethally irradiated CD45.1.1+ mice. After 6–8 weeks, bone marrow chimaeras were analysed for the expression of CD44, CD62L and IFNγ (c) and frequencies of CD44high CD62Llow effector/memory cells and CD44+ IFNγ+ cells (d) in splenic CD8+ T cells derived from wild-type or Mst1/2∆DC donor bone marrow cells (CD45.2.2+) or spike cells CD45.1.2+ (n = 5 mice per genotype). Numbers in quadrants indicate percentage of cells. Data are shown as mean and s.e.m. **P < 0.01; two-tailed unpaired Student’s t-test in a, b, d. Data summarize two (a, b) or three (c, d) independent experiments.Source Data.

  4. Extended Data Fig. 4 In vivo T cell responses in wild-type and Mst1/2∆DC mice challenged with tumour, pathogen or cognate antigen.

    a, b, Flow cytometry analysis of IFNγ expression (left) and frequencies of IFNγ+ cells (right) in CD8+ (a) and CD4+ (b) T cells from draining lymph node (DLN) and tumour tissues of wild-type and Mst1/2∆DC mice challenged with MC38 tumour cells (n = 7 for wild type, n = 5 for Mst1/2∆DC for DLN; n = 4 for wild type, n = 8 for Mst1/2∆DC for tumour tissues). c, Flow cytometry analysis of PD-1 expression on CD8+ (upper) and CD4+ (lower) T cells from DLN and tumour tissues of wild-type and Mst1/2∆DC mice challenged with MC38 tumour cells. d, Flow cytometry analysis of LAG3 and TIM3 expression of CD8+ (upper) and CD4+ (lower) T cells from DLN of wild-type and Mst1/2∆DC mice challenged with MC38 tumour cells. e, Flow cytometry of H-2Kb-OVA+ CD8+ T cells in blood from wild-type and Mst1/2∆DC mice infected with LM-OVA. f, Flow cytometry (left) and frequencies (right) of IFNγ+ and TNFα+ cells of PMA and ionomycin-stimulated CD8+ T cells in the blood from wild-type and Mst1/2∆DC mice infected with LM-OVA (n = 5 for wild type, n = 4 for Mst1/2∆DC). g, CFSE dilution of donor OT-I T cells in OVA-immunized mice. Numbers in quadrants or gates indicate percentage of cells. Data are shown as mean and s.e.m. *P < 0.05, **P < 0.01; two-tailed unpaired Student’s t-test in a, b, f. Data summarize two (ae, g) independent experiments.Source Data.

  5. Extended Data Fig. 5 Altered homeostasis of Mst1/2-deficient DCs.

    a, Detailed gating strategy for flow cytometry of splenic conventional DCs (cDC), CD8α+ cDC (CD8α+ CD11b), CD8α cDC (CD8α CD11b+) and pDC populations in wild-type and Mst1/2∆DC mice. MHC-II, MHC class II. b, Frequencies and cell numbers of splenic DC populations in wild-type and Mst1/2∆DC mice as gated in a (n = 5 mice per genotype). c, Flow cytometry analysis of CD40, CD70, CD80, CD86, H-2Kb, MHC-II, 4-1BBL, OX40L and PD-L1 expression on splenic CD8α+ and CD8α DCs as gated in a. d, Flow cytometry (left) of CD45.1.2+ bone marrow cell-derived and CD45.2.2+ wild-type or Mst1/2∆DC donor bone marrow cell-derived splenic cDC, CD8α+ cDC, CD8α cDC and pDC populations in mixed chimaeras and frequencies (right) of CD8α+ cDCs in total cDCs and pDCs in spleen derived from wild-type or Mst1/2∆DC donor bone marrow cells in mixed chimaeras (n = 5 mice per genotype). e, Normalized chimaerism for the indicated DC subsets in mixed bone marrow chimaeras. The percentage of indicated DC subsets was normalized by that of B cells from same mice. The chimaerism of the wild type was set as 1 (n = 5 mice per genotype). f, Flow cytometry analysis of donor (wild-type or Mst1/2∆DC, CD45.2.2+) and spike (CD45.1.2+) bone marrow cell percentages in the bone marrow mixture before transfer. Numbers in gates indicate percentage of cells. Data are shown as mean and s.e.m. *P < 0.05, **P < 0.01; two-tailed unpaired Student’s t-test in b, d, e. Data summarize four (ac) or three (d, e) independent experiments.Source Data.

  6. Extended Data Fig. 6 Homeostasis of DCs after deletion of Hippo pathway genes.

    a, Frequencies of splenic cDC, CD8α+ cDC, and pDC populations in wild-type (n = 9), Cd11ccreStk4fl/flStk3+/+ (n = 5), Cd11ccreStk4+/+Stk3fl/fl (n = 4), Cd11ccreStk4fl/+Stk3fl/fl (n = 5) and Cd11ccreStk4fl/flStk3fl/+ (n = 4) mice. b, c, Flow cytometry of splenic cDC, CD8α+ cDC, CD8α cDC and pDC populations in wild-type and Lats1/2∆DC (b), or Yap/Taz∆DC (c) mice. Numbers in gates indicate percentage of cells. Data summarize two (b), three (c) or eight (a) independent experiments.Source Data.

  7. Extended Data Fig. 7 Role of Mst1/2 in selectively programming functions of CD8α+ DCs and CD24high FLT3L-BMDCs to prime CD8 T cells.

    a, Frequency of CFSElow cells of donor OT-I T cells in wild-type (n = 5), Mst1/2∆DC (n = 5), Batf3−/−:wild-type (n = 5) or Batf3−/−:Mst1/2∆DC (n = 6) mixed chimaeras immunized with OVA. b, CFSE-labelled OT-I T cells (CD45.1+) were transferred to Β2m−/− mice followed by immunization one day later with OVA-pulsed CD8α+ or CD8α splenic DCs isolated from wild-type or Mst1/2∆DC mice (following DC expansion by FLT3L), and CFSE dilution of OT-I cells was examined three days after DC immunization. Shown are representative flow cytometry histograms of CFSE dilution and frequency of CFSElow cells in donor OT-I T cells (gated on CD45.1+CD45.2) from spleen of B2m−/− mice (n = 4 for Mst1/2-deficient CD8α DCs, n = 3 for all others). c, d, CD8α+ (c) or CD8α DCs (d) from wild-type and Mst1/2∆DC mice were fed with 0 or 50 μg/ml soluble OVA conjugated with FITC for 1 h at 37 °C or 4 °C. Cells were then collected and OVA uptake was evaluated by flow cytometry. Numbers in graphs indicate the mean fluorescence intensity of OVA-FITC. e, Flow cytometry (upper) and quantification (lower, n = 4 per genotype) of apoptotic CD8α+ or CD8α DCs examined by Annexin V (left) and active caspase 3 (right) staining in freshly isolated splenocytes from wild-type and Mst1/2∆DC mice. f, CD8α+ (upper) or CD8α DCs (lower) from wild-type and Mst1/2∆DC mice were cultured overnight for analysis of cell viability by 7-aminoactinomycin D (7-AAD) staining. Quantification of the percentage of 7-AAD-negative live cells is shown (n = 3 per genotype). g, Thymidine incorporation of OT-II T cells cultured with OVA protein- or OVA(323–339) peptide-pulsed CD8α+ or CD8α DCs (n = 13 derived from five mice for the Mst1/2-deficient CD8α+ DC/OVA(323–339) group, n = 15 derived from five mice for all other groups). hj, Thymidine incorporation of OT-I (left) or OT-II (right) T cells cultured with OVA protein-pulsed CD24high FLT3L-BMDCs (h), CD24low FLT3L-BMDCs (i), or GM-CSF-derived BMDCs (j) from wild-type and Mst1/2∆DC mice for 72 h (n = 14 from five mice for the Mst1/2-deficient GM-CSF-BMDC/OT-I group, n = 15 from five mice for all other groups). k, Relative thymidine incorporation of OT-I T cells cultured with OVA protein- or OVA(257–264) peptide-pulsed splenic CD8α+ DCs pre-treated with vehicle or Mst1/2 inhibitor (XMU-MP-1) for 4 h. Thymidine incorporation of OT-I T cells cultured with vehicle-treated DCs in each group was set as 1 (n = 3 per group). l, Relative thymidine incorporation of OT-I T cells cultured with OVA protein- or OVA(257–264) peptide-pulsed CD8α+ DCs from WT and Lats1/2∆DC mice (n = 14 derived from five mice for wild type, n = 15 derived from five mice for Lats1/2∆DC). Thymidine incorporation of OT-I T cells cultured with wild-type DCs in each group was set as 1. m, Relative thymidine incorporation of OT-I T cells cultured with OVA protein-pulsed wild-type or Yap/Taz∆DC splenic CD8α+ DCs that were pre-treated with vehicle or Mst1/2 inhibitor (n = 3 derived from two mice per genotype). Thymidine incorporation of OT-I T cells cultured with vehicle-treated DCs was set as 1. Numbers in gates indicate percentage of cells. Data are shown as mean and s.e.m. *P < 0.05, **P < 0.01; two-tailed unpaired Student’s t-test in a, b, el; two-way ANOVA in m. Data summarize two (ad, gj), three (e, f, k, l) or four (e) independent experiments.Source Data.

  8. Extended Data Fig. 8 Analysis of mitochondrial profiles of Mst1/2-, Lats1/2- or mTOR-deficient DCs.

    a, Functional annotations of upregulated metabolic pathways according to KEGG and Hallmark databases in CD8α+ DCs (compared to CD8α DCs) profiled using proteomics. b, ECAR of splenic CD8α+ and CD8α DCs. Oligo, oligomycin; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone. c, Flow cytometry analysis of mitochondrial mass and membrane potential of wild-type splenic CD8α+ and CD8α DCs using Mitotracker and TMRM (Tetramethylrhodamine, methyl ester) staining, respectively. Numbers in graph indicate the mean fluorescence intensity. d, Relative thymidine incorporation of OT-I T cells cultured with OVA protein- or OVA(257–264) peptide-pulsed CD8α+ and CD8α DCs pre-treated with vehicle or metabolic inhibitors. Values after culture with vehicle-treated DCs were set as 1. e, Thymidine incorporation of OT-I T cells cultured with OVA protein- or OVA(257–264) peptide-pulsed splenic CD8α+ DCs from wild-type and mTOR∆DC mice for 72 h (n = 12 from four mice per genotype). f, Flow cytometry analysis of mitochondrial mass and mitochondrial membrane potential of wild-type and Mst1/2∆DC splenic CD8α+ and CD8α DCs by Mitotracker and TMRM staining, respectively. Numbers in graph indicate the mean fluorescence intensity. g, Transmission electron microscopy analysis of mitochondria of splenic CD8α DCs from wild-type or Mst1/2∆DC mice. Arrows indicate mitochondria. h, Immunoblot analysis of expression of NDUFB8 (complex I), SDHB (complex II), UQCRC2 (complex III), MT-CO1 (complex IV) and ATP5A (complex V) in CD8α+ and CD8α DCs. i, Flow cytometry analysis of mitochondrial mass and mitochondrial membrane potential of wild-type and Lats1/2∆DC splenic CD8α+ and CD8α DCs by Mitotracker and TMRM staining, respectively. Numbers in graph indicate the mean fluorescence intensity. j, Immunoblot analysis of p-S6 and c-Myc protein in CD8α+ and CD8α DCs of wild-type and Mst1/2∆DC mice. k, Immunoblot analysis of NDUFB8 (complex I), SDHB (complex II), UQCRC2 (complex III), MT-CO1 (complex IV) and ATP5A (complex V) protein in CD8α+ and CD8α DCs of wild-type and mTOR∆DC mice. Data are shown as mean and s.e.m. *P < 0.05, **P < 0.01; one-way ANOVA in d; two-tailed unpaired Student’s t-test in e. Data summarize two (e, f, hk), three (b, d) or four (c) independent experiments.Source Data.

  9. Extended Data Fig. 9 Selective regulation of IL-12 signalling and expression by Mst1/2 in CD8α+ DCs.

    a, Venn diagram showing the overlap of top 28 upregulated (Mst1/2∆DC versus wild type) pathways by GSEA between CD8α+ and CD8α DCs. Briefly, transcriptional profiles of Mst1/2-deficient CD8α+ and CD8α DCs were compared to their respective wild-type counterparts by GSEA, and then upregulated pathways (FDR < 0.05) were identified in CD8α+ DCs (Mst1/2ΔDC versus wild type) and CD8α DCs (Mst1/2ΔDC versus wild type) and used to generate a Venn diagram. The top 28 upregulated pathways in Mst1/2-deficient CD8α+ and CD8α DCs (compared to their respective wild-type counterparts) were largely shared (24/28) between the two DC subsets. b, List of the significantly upregulated (FDR < 0.05) 24 pathways (out of 28) shared by Mst1/2-deficient CD8α+ and CD8α DCs (compared to their respective wild-type counterparts), as revealed by GSEA. c, List of the significantly downregulated (FDR < 0.05) pathways determined by GSEA (arranged according to FDR values) in Mst1/2-deficient CD8α+ DCs (versus wild-type cells). NES, normalized enrichment score. d, Il12b expression in wild-type CD8α+ and CD8α DCs. e, Relative IL-12 p40 cytokine concentration in the supernatant of lipopolysaccharide (LPS)-treated CD8α+ and CD8α DCs from wild-type and Mst1/2∆DC mice (n = 4 per genotype for CD8α+ DCs, n = 3 per genotype for CD8α DCs). IL-12 p40 cytokine concentration of wild-type DCs was set as 1. f, Real-time PCR analysis of Il12b mRNA expression in wild-type splenic CD8α+ and CD8α DCs treated with vehicle or Mst1/2 inhibitor (XMU-MP-1) (n = 4 per treatment). g, Il12b expression in FLT3L-BMDCs (n = 4 for wild-type CD24high BMDCs, n = 5 for other groups). h, i, Real-time PCR analysis of Il1a, Il1b, Il6, Il10 and Ifnb1 mRNA expression in splenic CD8α+ (h) and CD8α (i) DCs from wild-type and Mst1/2ΔDC mice (n = 4 mice per genotype). j, k, Frequencies of CD44high CD62Llow (j) and CD44+ IFNγ+ (k) cells in splenic CD4+ and CD8+ T cells from wild-type (n = 5), Mst1/2∆DC (n = 5), Il12a−/− (n = 6) and Mst1/2∆DC Il12a−/− (n = 6) mice. Wild-type and Mst1/2ΔDC groups were the same as those shown in Fig. 1d, e. Data are shown as mean and s.e.m. *P < 0.05, **P < 0.01; unpaired Student’s t-test in d, fi; two-tailed paired Student’s t-test in e; one-way ANOVA in j, k. Data summarize two (d, g), three (e), four (j, k) or five (f) independent experiments.Source Data.

  10. Extended Data Fig. 10 Mst/Hippo signalling integrates mitochondrial metabolism and non-canonical NF-κB/IL-12 signalling in CD8α+ DCs.

    a, Relative thymidine incorporation of OT-I T cells cultured with OVA protein- or OVA(257–264) peptide-pulsed splenic CD8α+ DCs (left) or CD8α DCs (right) from wild-type and Il12a−/− mice (n = 9 derived from three mice for wild type, n = 12 derived from four mice for Il12a−/−). Thymidine incorporation of OT-I T cells cultured with wild-type DCs in each group was set as 1. b, Real-time PCR analysis of Nfkb2 (left) or Relb (right) mRNA expression in splenic CD8α+ DCs from wild-type and Mst1/2∆DC mice (n = 3 mice per population). c, NF-κB2 and RelB expression in control- or NIK(Δ78–84)-transduced wild-type CD11c+ B220 FLT3L-BMDCs. d, Flow cytometry analysis of mitochondrial membrane potential of wild-type or Mst1/2-deficient CD24high FLT3L-BMDCs transduced with control (left) or NIK(∆78–84) (right) virus. Numbers in graph indicate the mean fluorescence intensity. e, Thymidine incorporation of OT-I T cells cultured with OVA protein-pulsed wild-type or Mst1/2-deficient CD24high FLT3L-BMDCs transduced with control or NIK(∆78–84) virus (n = 6 derived from three mice for wild-type/control group, n = 8 derived from three mice for Mst1/2∆DC/control group, n = 4 derived from three mice per genotype for NIK(∆78–84) group). f, FLT3L-expanded splenic CD8α+ DC lysate was immunoprecipitated with anti-Mst1 antibody and blotted with anti-Traf3. Mst1 blot was from the same experiment as Fig. 3f. g, Immunoblot analysis of Traf3 protein in splenic CD8α+ and CD8α DCs from wild-type and Mst1/2∆DC mice. h, Real-time PCR analysis of Il12b expression in wild-type CD8α+ DCs treated with vehicle, metformin or oligomycin as indicated in figures (n = 3 per group). Il12b expression of vehicle-treated DCs was set as 1. i, Flow cytometry analysis of mitochondrial membrane potential (upper) and mitochondrial mass (lower) of different concentrations of IL-12-treated wild-type splenic CD8α+ DCs by TMRM (tetramethylrhodamine, methyl ester) and Mitotracker staining. Numbers in graph indicate the mean fluorescence intensity. j, Flow cytometry analysis of mitochondrial membrane potential and mitochondrial mass of wild-type and Il12a−/− splenic CD8α+ DCs by TMRM and Mitotracker staining. k, Flow cytometry analysis of mitochondrial membrane potential and mitochondrial mass of wild-type and NIK-deficient (Map3k14−/−) splenic CD8α+ DCs by TMRM and Mitotracker staining. l, Immunoblot analysis of p-Lats1/2, p-Yap and p-Mst1/2 proteins in wild-type splenic CD8α+ DCs treated with FLT3L for the indicated times. m, CD8α+ DC number from spleen of wild-type and Mst1/2∆DC mice treated with or without FLT3L for 10 days. n, Flow cytometry analysis of mitochondrial mass of human CD141+ DCs treated with vehicle or Mst1/2 inhibitor (XMU-MP-1) by Mitotracker staining. o, Real-time PCR analysis of Il12b mRNA expression in human CD141+ (equivalent to mouse CD8α+ DCs) and CD1c+ DCs (equivalent to mouse CD8α DCs) treated with vehicle or Mst1/2 inhibitor (XMU-MP-1) (n = 5 for CD141+ DC, n = 4 for CD1c+ DC). Data are shown as mean and s.e.m. *P < 0.05, **P < 0.01; two-tailed unpaired Student’s t-test in a, b, m, o; two-way ANOVA in e. Data summarize two (a, dg, i, j, l, n, o) or three (h, k, m) independent experiments. p, Brief schematics of non-canonical Hippo signalling in orchestrating CD8α+ DC function. Mst/Hippo signalling integrates metabolic and IL-12 cytokine signalling in CD8α+ DCs through controlling mitochondrial dynamics and non-canonical NF-κB signalling. This regulation is independent of canonical Hippo signalling in organ size control and tumour suppression.Source Data.

Supplementary information

  1. Supplementary Figure 1

    The uncropped western blot images with size marker indications.

  2. Reporting Summary

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https://doi.org/10.1038/s41586-018-0177-0

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