Journal: Science (New York, N.Y.)
Article Title: Mitochondrial metabolism and signaling direct dendritic cell function in antitumor immunity
doi: 10.1126/science.adv6582
Figure Lengend Snippet: ( A ) B16-OVA tumor-bearing WT and Opa1 DDC mice were euthanized at day 14 after tumor inoculation ( n = 6 each group). Quantification of the geometric mean fluorescence intensity (gMFI) of H-2K b -SIINFEKL complex in intratumoral cDC1s and cDC2s. ( B ) CellTrace Violet ™ (CTV)-labeled naïve OT-I cells were adoptively transferred intravenously (i.v.) into WT or Opa1 ΔDC mice, followed by i.v. immunization with 20 μg OVA protein 24 hours later. After 3 days, the frequency of CTV lo (proliferated) OT-I cells in spleens was quantified by flow cytometry analysis ( n = 8 for WT; 10 for Opa1 ΔDC ). ( C ) Sort-purified splenic WT or OPA1-deficient cDC1s were pulsed with heat-inactivated OVA-expressing Listeria monocytogenes (HKLM-OVA) in complete IMDM medium for 4 hours, followed by their irradiation and coculture with OT-I cells at a ratio of 1:10. Thymidine incorporation of OT-I cells was measured 72 hours later ( n = 12 each group). ( D ) Sort-purified splenic WT or OPA1-deficient cDC1s were pulsed with B16F10 tumor cell lysate in complete IMDM medium for 4 hours, followed by their irradiation and coculture with pmel cells at a ratio of 1:10. Thymidine incorporation of pmel cells was measured 72 hours later ( n = 4 for cDC1s; 6 for cDC2s). ( E ) ATAC-seq analysis of sort-purified splenic cDC1s from WT and Opa1 ΔDC mice ( n = 4 per genotype). Transcription factor footprinting analysis was performed by comparing Opa1 ΔDC versus WT splenic cDC1s, and the transcription factors with more 1,000 binding sites were selected and then ranked by their Z-scores. Blue dots and red dots indicate transcription factors predicted to have decreased and increased activity, respectively, and NRF1 is identified as the most downregulated transcription factor. ( F ) Immunoblot analysis of NRF1 expression in cDC1s from WT and Opa1 ΔDC mice. ACTB was used as loading control (left). Quantification of the relative expression of NRF1 in cDC1s from WT and Opa1 ΔDC mice (right). ( G ) Venn diagram showing the significant overlap between downregulated genes in OPA1-deficient cDC1s versus WT cDC1s compared to putative NRF1 target genes (left). Functional enrichment analysis of the 188 overlapped genes, with the top downregulated Hallmark OXPHOS pathway labeled in blue color (right). ( H ) Quantification of the basal oxygen consumption rate (OCR) in splenic cDC1s expressing sgNTC or sg Nrf1 , which were sort-purified from “retrogenic mice” bearing B16-Flt3L tumors (for DC expansion) at day 9 after tumor inoculation, as measured by Seahorse metabolic flux assay; see also fig. S7G ( n = 9 for sgNTC; 6 for sg Nrf1 ). ( I ) Thymidine incorporation of OT-I cells cocultured with OVA protein-pulsed (left) or OVA 257–264 peptide-pulsed (right) splenic cDC1s expressing sgNTC or sg Nrf1 (isolated from the B16-Flt3L tumor-bearing “retrogenic” mice for DC expansion, see also fig. S7G ) for 72 hours ( n = 8 for sgNTC for both OVA and OVA 257–264 peptide; 5 for sg Nrf1 in response to OVA; 4 for sg Nrf1 in response to OVA 257–264 peptide). ( J ) “Retrogenic” mice bearing WT or OPA1-deficient cDC1s that overexpress empty vector or NRF1 protein were generated and injected with B16-Flt3L tumors (for DC expansion), followed by sort purification of cDC1s 9 days later (see also fig. S7G ). WT or OPA1-deficient cDC1s overexpressing empty vector or NRF1 were treated for 2 hours (during OVA protein pulse) with or without 500 nM rotenone (ETC complex I inhibitor), 1 mM 3-NPA (ETC complex II inhibitor), 0.5 μM antimycin A (ETC complex III inhibitor) or 1 μM oligomycin (ETC complex V inhibitor). OT-I cell proliferation was assessed after 72 hours ( n = 3 for OPA1-deficient cDC1 with antimycin A treatment; 4 for remaining groups). ( K ) Heatmap showing the expression of putative NRF1 targets within in the Hallmark OXPHOS pathway that were downregulated in OPA1-deficient cDC1s compared to WT cells [as shown in (G); ranked by row z-score] ( n = 3 for WT, 4 for Opa1 ΔDC ). ( L ) Immunoblot analysis of the expression of NDUFB8 (ETC complex I; CI), SDHB (ETC complex II; CII), UQCRC2 (ETC complex III; CIII), MTCO1 (ETC complex IV; CIV) and ATP5A (ETC complex V; CV) in splenic cDC1s from WT and Opa1 ΔDC mice. ACTB was used as loading control. Densitometric quantification of NDUFB8 was performed and normalized to ACTB expression. The numbers show the relative NDUFB8 values compared to WT cDC1s. ( M ) Thymidine incorporation of OT-I cells cocultured with OVA protein-pulsed (left) or OVA 257–264 peptide-pulsed (right) splenic cDC1s expressing sgNTC or sg Ndufaf1 (ETC complex I assembly factor; CI) (isolated from B16-Flt3L tumor-bearing “retrogenic” mice for DC expansion, see also fig. S7G ) for 72 hours ( n = 8 each group). ( N ) Thymidine incorporation of OT-I cells cultured with OVA protein-pulsed (left) or OVA 257–264 peptide-pulsed (right) splenic cDC1s expressing sgNTC, sg Sdhb (component of ETC complex II; CII), sg Uqcrq (component of ETC complex III; CIII) or sg Atpaf2 (ETC complex V assembly factor; CV) (isolated from B16-Flt3L tumor-bearing “retrogenic mice” for DC expansion, see also fig. S7G ) for 72 hours ( n = 5 for cDC1 with sg Uqcrq or sg Atpaf2 cultured with OVA protein; 6 for all other groups). ( O ) Thymidine incorporation of OT-I cells cocultured with OVA protein-pulsed splenic cDC1s from WT and Opa1 ΔDC mice that were pretreated for 2 hours (during antigen pulse) with or without 500 nM rotenone, 1 mM 3-NPA, 0.5 μM antimycin A or 1 μM oligomycin. OT-I cell proliferation was assessed after 72 hours ( n = 4 per group). ( P ) Thymidine incorporation of OT-I cells cocultured with OVA protein-pulsed splenic WT or OPA1-deficient cDC1s expressing sgNTC or sg Ndufaf1 , which were sort-purified from “retrogenic mice” bearing B16-Flt3L tumors (for DC expansion) at day 9 after tumor inoculation (as described in fig. S7G ) ( n = 4 for WT cDC1s expressing sg Ndufaf1 ; 5 for WT cDC1s expressing sgNTC or OPA1-deficient cDC1s expressing sg Ndufaf1 ; 6 for OPA1-deficient cDC1s expressing sgNTC). ( Q ) Thymidine incorporation of OT-I cells cocultured with OVA protein-pulsed splenic cDC1s expressing sgNTC or sg Nrf1 (isolated from B16-Flt3L tumor-bearing “retrogenic mice” for DC expansion, see also fig. S7G ) that were pretreated for 2 hours (during antigen pulse) with or without 500 nM rotenone, 1 mM 3-NPA, 0.5 μM antimycin A or 1 μM oligomycin. OT-I cell proliferation was assessed after 72 hours ( n = 5 for sgNTC, 4 for sg Nrf1 ). Data are shown as mean ± s.e.m. in (A to D, F, H to J, and M to Q). Two-way ANOVA in (A, C, D, J, O to Q), two-tailed unpaired Student’s t -test in (B, F, H, I and M) or one-way ANOVA in (N). Data are representative of two (C, D, H, J, and L, N to Q) or at least three (A, B, F, I and M) independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. NS, not significant. Numbers in plots represent percentages of cell in gates (B).
Article Snippet: For in vitro assays, cDC1s were sorted from spleen or tumor, pulsed with 200 μg/ml OVA protein (Low Endo, LS003059 , Worthington) or 250 pg/ml OVA 257–264 peptide (vac-sin, InvivoGen) for 2 hours, washed twice with IMDM complete medium and irradiated (3,000 rad), and then cocultured with naïve (CD44 lo CD62L hi ) OT-I cells for three days.
Techniques: Immunopeptidomics, Fluorescence, Labeling, Flow Cytometry, Purification, Expressing, Irradiation, Footprinting, Binding Assay, Activity Assay, Western Blot, Control, Functional Assay, Flux Assay, Isolation, Plasmid Preparation, Generated, Injection, Cell Culture, Two Tailed Test
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![( A ) <t>B16-OVA</t> tumor-bearing WT and Opa1 DDC mice were euthanized at day 14 after tumor inoculation ( n = 6 each group). Quantification of the geometric mean fluorescence intensity (gMFI) of H-2K b -SIINFEKL complex in intratumoral cDC1s and cDC2s. ( B ) CellTrace Violet ™ (CTV)-labeled naïve OT-I cells were adoptively transferred intravenously (i.v.) into WT or Opa1 ΔDC mice, followed by i.v. immunization with 20 μg OVA protein 24 hours later. After 3 days, the frequency of CTV lo (proliferated) OT-I cells in spleens was quantified by flow cytometry analysis ( n = 8 for WT; 10 for Opa1 ΔDC ). ( C ) Sort-purified splenic WT or OPA1-deficient cDC1s were pulsed with heat-inactivated OVA-expressing Listeria monocytogenes (HKLM-OVA) in complete IMDM medium for 4 hours, followed by their irradiation and coculture with OT-I cells at a ratio of 1:10. Thymidine incorporation of OT-I cells was measured 72 hours later ( n = 12 each group). ( D ) Sort-purified splenic WT or OPA1-deficient cDC1s were pulsed with B16F10 tumor cell lysate in complete IMDM medium for 4 hours, followed by their irradiation and coculture with pmel cells at a ratio of 1:10. Thymidine incorporation of pmel cells was measured 72 hours later ( n = 4 for cDC1s; 6 for cDC2s). ( E ) ATAC-seq analysis of sort-purified splenic cDC1s from WT and Opa1 ΔDC mice ( n = 4 per genotype). Transcription factor footprinting analysis was performed by comparing Opa1 ΔDC versus WT splenic cDC1s, and the transcription factors with more 1,000 binding sites were selected and then ranked by their Z-scores. Blue dots and red dots indicate transcription factors predicted to have decreased and increased activity, respectively, and NRF1 is identified as the most downregulated transcription factor. ( F ) Immunoblot analysis of NRF1 expression in cDC1s from WT and Opa1 ΔDC mice. ACTB was used as loading control (left). Quantification of the relative expression of NRF1 in cDC1s from WT and Opa1 ΔDC mice (right). ( G ) Venn diagram showing the significant overlap between downregulated genes in OPA1-deficient cDC1s versus WT cDC1s compared to putative NRF1 target genes (left). Functional enrichment analysis of the 188 overlapped genes, with the top downregulated Hallmark OXPHOS pathway labeled in blue color (right). ( H ) Quantification of the basal oxygen consumption rate (OCR) in splenic cDC1s expressing sgNTC or sg Nrf1 , which were sort-purified from “retrogenic mice” bearing B16-Flt3L tumors (for DC expansion) at day 9 after tumor inoculation, as measured by Seahorse metabolic flux assay; see also fig. S7G ( n = 9 for sgNTC; 6 for sg Nrf1 ). ( I ) Thymidine incorporation of OT-I cells cocultured with OVA protein-pulsed (left) or <t>OVA</t> <t>257–264</t> peptide-pulsed (right) splenic cDC1s expressing sgNTC or sg Nrf1 (isolated from the B16-Flt3L tumor-bearing “retrogenic” mice for DC expansion, see also fig. S7G ) for 72 hours ( n = 8 for sgNTC for both OVA and OVA 257–264 peptide; 5 for sg Nrf1 in response to OVA; 4 for sg Nrf1 in response to OVA 257–264 peptide). ( J ) “Retrogenic” mice bearing WT or OPA1-deficient cDC1s that overexpress empty vector or NRF1 protein were generated and injected with B16-Flt3L tumors (for DC expansion), followed by sort purification of cDC1s 9 days later (see also fig. S7G ). WT or OPA1-deficient cDC1s overexpressing empty vector or NRF1 were treated for 2 hours (during OVA protein pulse) with or without 500 nM rotenone (ETC complex I inhibitor), 1 mM 3-NPA (ETC complex II inhibitor), 0.5 μM antimycin A (ETC complex III inhibitor) or 1 μM oligomycin (ETC complex V inhibitor). OT-I cell proliferation was assessed after 72 hours ( n = 3 for OPA1-deficient cDC1 with antimycin A treatment; 4 for remaining groups). ( K ) Heatmap showing the expression of putative NRF1 targets within in the Hallmark OXPHOS pathway that were downregulated in OPA1-deficient cDC1s compared to WT cells [as shown in (G); ranked by row z-score] ( n = 3 for WT, 4 for Opa1 ΔDC ). ( L ) Immunoblot analysis of the expression of NDUFB8 (ETC complex I; CI), SDHB (ETC complex II; CII), UQCRC2 (ETC complex III; CIII), MTCO1 (ETC complex IV; CIV) and ATP5A (ETC complex V; CV) in splenic cDC1s from WT and Opa1 ΔDC mice. ACTB was used as loading control. Densitometric quantification of NDUFB8 was performed and normalized to ACTB expression. The numbers show the relative NDUFB8 values compared to WT cDC1s. ( M ) Thymidine incorporation of OT-I cells cocultured with OVA protein-pulsed (left) or OVA 257–264 peptide-pulsed (right) splenic cDC1s expressing sgNTC or sg Ndufaf1 (ETC complex I assembly factor; CI) (isolated from B16-Flt3L tumor-bearing “retrogenic” mice for DC expansion, see also fig. S7G ) for 72 hours ( n = 8 each group). ( N ) Thymidine incorporation of OT-I cells cultured with OVA protein-pulsed (left) or OVA 257–264 peptide-pulsed (right) splenic cDC1s expressing sgNTC, sg Sdhb (component of ETC complex II; CII), sg Uqcrq (component of ETC complex III; CIII) or sg Atpaf2 (ETC complex V assembly factor; CV) (isolated from B16-Flt3L tumor-bearing “retrogenic mice” for DC expansion, see also fig. S7G ) for 72 hours ( n = 5 for cDC1 with sg Uqcrq or sg Atpaf2 cultured with OVA protein; 6 for all other groups). ( O ) Thymidine incorporation of OT-I cells cocultured with OVA protein-pulsed splenic cDC1s from WT and Opa1 ΔDC mice that were pretreated for 2 hours (during antigen pulse) with or without 500 nM rotenone, 1 mM 3-NPA, 0.5 μM antimycin A or 1 μM oligomycin. OT-I cell proliferation was assessed after 72 hours ( n = 4 per group). ( P ) Thymidine incorporation of OT-I cells cocultured with OVA protein-pulsed splenic WT or OPA1-deficient cDC1s expressing sgNTC or sg Ndufaf1 , which were sort-purified from “retrogenic mice” bearing B16-Flt3L tumors (for DC expansion) at day 9 after tumor inoculation (as described in fig. S7G ) ( n = 4 for WT cDC1s expressing sg Ndufaf1 ; 5 for WT cDC1s expressing sgNTC or OPA1-deficient cDC1s expressing sg Ndufaf1 ; 6 for OPA1-deficient cDC1s expressing sgNTC). ( Q ) Thymidine incorporation of OT-I cells cocultured with OVA protein-pulsed splenic cDC1s expressing sgNTC or sg Nrf1 (isolated from B16-Flt3L tumor-bearing “retrogenic mice” for DC expansion, see also fig. S7G ) that were pretreated for 2 hours (during antigen pulse) with or without 500 nM rotenone, 1 mM 3-NPA, 0.5 μM antimycin A or 1 μM oligomycin. OT-I cell proliferation was assessed after 72 hours ( n = 5 for sgNTC, 4 for sg Nrf1 ). Data are shown as mean ± s.e.m. in (A to D, F, H to J, and M to Q). Two-way ANOVA in (A, C, D, J, O to Q), two-tailed unpaired Student’s t -test in (B, F, H, I and M) or one-way ANOVA in (N). Data are representative of two (C, D, H, J, and L, N to Q) or at least three (A, B, F, I and M) independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. NS, not significant. Numbers in plots represent percentages of cell in gates (B).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9015/pmc13109015/pmc13109015__nihms-2161853-f0002.jpg)