Journal: mBio

Article Title: Wolbachia uses ankyrin repeats to target specific fly proteins

doi: 10.1128/mbio.00172-26

Figure Lengend Snippet: WARP434 and WARP754 induce toxic phenotypes in the fly upon expression. ( A ) Schematic of constructs generated for expression in the fly, both full-length WARPs and those missing the ankyrin repeat domains. ( B ) Wild-type tissues (across top) compared to expression of constructs suggests that the ankyrin repeat domain-containing regions of WARPs are required for toxicity. Arrowheads point to sex combs and genitalia in those relevant panels.

Article Snippet: Rabbit polyclonal antibody sera against both full-length purified proteins were generated (Cocalico Biologicals, Inc.) and used separately for immunochemistry (see below).

Techniques: Expressing, Construct, Generated

Journal: eLife

Article Title: GTPase-activating protein DLC1 spatio-temporally regulates Rho signaling

doi: 10.7554/eLife.90305

Figure Lengend Snippet: ( A ) Example fields of view of tracked WT and DLC1 KO cells. 20,000 REF fibroblasts expressing a histone H2B-miRFP marker were seeded in a 24-well plate well coated with fibronectin and stimulated with 20 ng/ml PDGF, imaged for 12 hr at 15 min interval. Tracking was performed using Stardist 37 on the H2B image. DLC1 KO cells display a large subpopulation of cells that remain extremely stationary. ( B ) Mean Square displacements (MSDs) for different time intervals, WT cells move more than DLC1 KO cells for all possible lag times. The plot shows directionality (exponential bit between 0.25 and 1.5 hr). For times longer than 2 hr, the curve is flat, indicating that migration has characteristics of random walk at these timescales. Thick lines: mean, thin lines: standard deviation. A dotted gray line shows delta t chosen for figure C. ( C ) Distribution of velocities calculated as µm/hr from Root-MSD. We again observe the bimodal distribution in the DLC1 KO cell with a big subgroup of the cells moving extremely slowly (5 µm/hr). Thick lines are mean and extrema. Two-sided t-test: (statistic = 25.1, p-value = 1.6e-131).

Article Snippet: Glass-bottom well plates (Celvis) were coated with 5 μg/ml of human plasma fibronectin purified protein (Merck) for 1 hr at room temperature.

Techniques: Expressing, Marker, Migration, Standard Deviation

Journal: eLife

Article Title: GTPase-activating protein DLC1 spatio-temporally regulates Rho signaling

doi: 10.7554/eLife.90305

Figure Lengend Snippet: Effect of DLC1 overexpression on F-actin. REF52 cells stably expressing Lifeact-mCherry were transfected with mCherry-DLC1 or mCherry plasmids, allowed to spread for 12 hr on fibronectin-coated coverslips, and imaged. Images are shown in ibw contrast. Large mCherry-DLC1 pool in the cytosol levels documents its overexpression. Note how DLC1 overexpression leads to loss of contractile F-actin structures.

Article Snippet: Glass-bottom well plates (Celvis) were coated with 5 μg/ml of human plasma fibronectin purified protein (Merck) for 1 hr at room temperature.

Techniques: Over Expression, Stable Transfection, Expressing, Transfection

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 ) Flow cytometry plots (left) of TMRM (for mitochondrial membrane potential) and MitoTracker ™ Green (MG; for mitochondrial mass) staining in cDC1s isolated from B16-OVA tumors at day 10–12 after tumor inoculation. Based upon TMRM and MG co-staining, two subpopulations of cDC1s were labeled as [TMRM/MG] hi and [TMRM/MG] lo cells as indicated. Quantification (right) of the percentage of each of subpopulation among intratumoral cDC1s ( n = 26 per group). ( B to D ) Sort-purified intratumoral cDC1 subpopulations from B16-OVA (B), EO771 (C) or HCC (D) tumors were pulsed with OVA protein in complete IMDM medium for 2 hours, followed by their irradiation and coculture with OT-I at a ratio of 1:10 for 72 hours. OT-I cell proliferation was measured by thymidine incorporation ( n = 5 per group in B; n = 5 for [TMRM/MG] lo cDC1s and 9 for [TMRM/MG] hi cDC1s in C; n = 4 for [TMRM/MG] lo cDC1s and 6 for [TMRM/MG] hi cDC1s in D). ( E ) Uniform manifold approximation and project (UMAP) plot of cDC1s from merged samples of published scRNA-seq datasets (E-MTAB-8107/6149/6653) of human colorectal, ovarian and lung tumors. For the UMAP, the activity score of our in-house generated cDC1 [TMRM/MG] hi UP signature (see Methods) is depicted. Each dot corresponds to an individual cDC1 and is color-coded based on high (red) or low (blue) activity score of the cDC1 [TMRM/MG] hi UP signature. Circles indicate discrete subpopulations of human cDC1s from the datasets based on the activity of cDC1 [TMRM/MG] hi UP signature. ( F ) WT mice were inoculated with B16-OVA melanoma cells. At day 12 after tumor challenge, intratumoral and splenic cDC1s were isolated for proteomics analysis ( n = 3 per group). Pathway enrichment analysis was performed based on the differentially expressed proteins (log 2 FC > 0.5 and false discovery rate (FDR) < 0.01) in cDC1s from the tumor versus spleen using mitochondria-related pathways from the Mouse MitoCarta3.0 database. Cristae formation (labeled in red color) ranks as the second most upregulated mitochondria-associated pathway. ( G ) Electron microscopy (EM) analysis (left) of cristae (arrows) in intratumoral cDC1s derived from B16-OVA tumor-bearing mice at 12 days after tumor inoculation. Scale bars: 0.5 μm. Quantification (right) of cristae number or length per mitochondrion ( n = 50 mitochondria for splenic cDC1s; 40 mitochondria for intratumoral cDC1s). ( H ) EM analysis of mitochondria in cDC1s derived from B16-OVA tumors at day 12. The second and fourth panels are zoomed in inlets of the first and third panels, respectively. Scale bars: 1 μm. ( I ) Flow cytometry plots (left) of TMRM and MitoTracker Green ™ (MG) staining in intratumoral cDC1s isolated from B16-OVA tumor-bearing WT and Opa1 ΔDC mice at 10 days after tumor inoculation. Quantification (right) of the percentages of [TMRM/MG] hi and [TMRM/MG] lo cells (based on TMRM and MG co-staining as indicated) among intratumoral cDC1s ( n = 6 per group). ( J ) 0.5 × 10 6 B16-OVA cells were inoculated in WT and Opa1 ΔDC mice ( n = 8 per group). Tumor growth was monitored. ( K and L ) WT ( n = 8) and Opa1 ΔDC ( n = 5) mice received AKT and NRAS G12V oncogenic vectors via hydrodynamic tail-vein injection (HDI) to induce liver tumorigenesis, and liver tumor burden was analyzed on day 29 after HDI. Representative images of liver tissue (K, left), and quantification of liver weight (K, middle) or the ratio of liver/body weight (K, right). Representative hematoxylin and eosin staining of liver tissue (scale bars: 100 μm) (L). ( M and N ) WT and Opa1 ΔDC mice were inoculated with B16-OVA cells and euthanized at day 14 after tumor inoculation ( n = 7 per group). Quantification of the frequencies and numbers (normalized to tumor weight) of intratumoral CD8 + T cells (CD8 + TCRb + ), non-Treg CD4 + T cells (CD4 + TCRb + FOXP3 − ) and Treg cells (CD4 + TCRb + FOXP3 + ) (M). Quantification of the frequencies and numbers (normalized to tumor weight) of IFNg + (first and second panels) and granzyme B (GZMB) + (third and fourth panels) cells among intratumoral CD8 + T cells, after stimulation with phorbol myristate acetate (PMA) and ionomycin in the presence of GolgiSTOP for 4 hours (N). ( O ) B16-OVA tumor growth curves in WT ( n = 10) and Xcr1 cre/+ Opa1 fl/fl ( n = 12) mice. ( P ) MC38 tumor growth curves in WT ( n = 7) and Xcr1 cre/+ Opa1 fl/fl ( n = 8) mice. ( Q ) LLC tumor growth curves in WT and Xcr1 cre/+ Opa1 fl/fl ( n = 6 per group) mice. Data are shown as mean ± s.e.m. in (A to D, G, I to K, and M to Q). Two-tailed unpaired Student’s t -test in (B to D, G, K and N), two-way ANOVA in (I, J, M and O to Q). Data are representative of one (H), two (C, D, I, K, L and Q) or at least three (B, J, and M to P), or pooled from four (A) independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. NS, not significant. Numbers indicate percentages of cells in gates (A and I).

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: Activation Assay, Flow Cytometry, Membrane, Staining, Isolation, Labeling, Purification, Irradiation, Activity Assay, Generated, Electron Microscopy, Derivative Assay, Injection, Two Tailed Test

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

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 ) ATP abundance (upper), and the ratios of ADP to ATP (middle) and AMP to ATP (lower) were measured in splenic cDC1s from WT and Opa1 ΔDC mice ( n = 3 per group). ( B and C ) Immunoblot analysis of the indicated proteins in splenic cDC1s that were isolated from WT and Opa1 ΔDC mice (B) or in splenic cDC1s expressing sgNTC or sg Nrf1 isolated from “retrogenic” mice bearing B16-Flt3L tumors (for DC expansion) at day 9 after tumor inoculation; see also fig. S7G (C). ACTB was used as a loading control. Densiometric quantification of p-AMPK, p-ULK1, P62, or LC3-II was performed and normalized to total protein (for p-AMPK and p-ULK1), ACTB (for P62) or LC3-I (for LC3-II) expression. Numbers show the relative values of each of protein compared to WT cDC1s (B) or cDC1s expressing sgNTC (C). ( D ) Splenic cDC1s from WT and Opa1 ΔDC mice were treated with or without chloroquine (CQ) overnight in vitro ( n = 4 per group). Quantification of the geometric mean fluorescence intensity (gMFI) of surface MHC-I on WT and OPA1-deficient cDC1s without CQ treatment (left). Quantification of the ratio of MHC-I gMFI in CQ-treated (+CQ) compared to untreated (−CQ) splenic cDC1s from WT and Opa1 ΔDC mice (right). ( E ) Sort-purified splenic cDC1s from WT and Opa1 ΔDC mice were pulsed with DQ-OVA for the indicated times with or without CQ, followed by flow cytometry analysis. Quantification of the percentage of FITC + cells (indicative of DQ-OVA degradation) among cDC1s is shown ( n = 3 per group). ( F ) “Retrogenic” mice were generated by transducing LSK cells from Cas9-expressing WT and Opa1 ΔDC mice with sgNTC or sg Atg5 , followed by their inoculation with B16-Flt3L tumors for DC expansion (see fig. S7G ). cDC1s were isolated from these mice 9 days later. Quantification of surface MHC-I gMFI on splenic WT or OPA1-deficient cDC1s expressing sgNTC or sg Atg5 isolated from the above described “retrogenic” mice ( n = 4 for WT and OPA1-deficient cDC1 with sgNTC groups; 3 for WT+sg Atg5 and Opa1 ΔDC +sg Atg5 groups). ( G ) Thymidine incorporation of OT-I cells cocultured with OVA protein-pulsed WT or OPA1-deficient cDC1s expressing sgNTC or sg Atg5 (generated as described in F) for 72 hours ( n = 3 for Opa1 ΔDC +sg Atg5 group; 6 for all other groups). ( H ) NAD + /NADH ratio was measured in splenic cDC1s from WT and Opa1 ΔDC mice ( n = 6 per group). ( I ) “Retrogenic” mice bearing WT or OPA1-deficient cDC1s that overexpress empty vector or mito- Lb NOX protein were generated and injected with B16-Flt3L tumors for DC expansion, followed by sort purification of splenic cDC1s 9 days later (see also fig. S7G ). Thymidine incorporation of OT-I cells cocultured with OVA protein-pulsed WT or OPA1-deficient cDC1s overexpressing empty vector or mito- Lb NOX for 72 hours ( n = 3 for WT+mito- Lb NOX and Opa1 ΔDC +mito- Lb NOX; 6 for WT+Vector and Opa1 ΔDC +Vector). ( J ) Seahorse metabolic flux analysis of oxygen consumption rate (OCR) of splenic cDC1s from WT, Opa1 ΔDC , Dnm1l ΔDC or Opa1 / Dnm1l ΔDC mice. Cells were treated with the indicated mitochondrial inhibitors (Oligo, oligomycin; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; and Rot, rotenone; upper). Basal OCR (before Oligo treatment; middle) and maximal OCR (after FCCP treatment; lower) of splenic cDC1s isolated from WT, Opa1 ΔDC , Dnm1l ΔDC or Opa1 / Dnm1l ΔDC mice ( n = 6 for WT and Opa1 ΔDC groups; 9 for Dnm1l ΔDC and Opa1 / Dnm1l ΔDC groups). ( K ) Immunoblot analysis of NRF1 expression in cDC1s from WT, Opa1 ΔDC , Dnm1l ΔDC or Opa1 / Dnm1l ΔDC mice. ACTB was used as loading control (upper). Quantification of the relative expression of NRF1 in cDC1s from WT, Opa1 ΔDC , Dnm1l ΔDC or Opa1 / Dnm1l ΔDC mice (lower). ( L ) B16-OVA tumor growth curves in WT ( n = 6), Opa1 ΔDC ( n = 4), Dnm1l ΔDC ( n = 6) and Opa1 / Dnm1l ΔDC ( n = 5) mice. ( M ) MC38 tumor growth curves in WT ( n = 11), Opa1 ΔDC ( n = 8), Dnm1l ΔDC ( n = 8) and Opa1 / Dnm1l ΔDC ( n = 5) mice. Data are shown as mean ± s.e.m. in (A, D to M). Two-tailed unpaired Student’s t -test in (A, D and H), two-way ANOVA in (E, L and M) or one-way ANOVA in (F, G, I, J and K). Data are representative of two (A to D, F, G, L and M) or at least three (E, I, J and K), or pooled from two (H) independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. NS, not significant.

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: Inhibition, Functional Assay, Western Blot, Isolation, Expressing, Control, In Vitro, Fluorescence, Purification, Flow Cytometry, Generated, Plasmid Preparation, Injection, Two Tailed Test

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 ) Sort-purified intratumoral cDC1s from B16-OVA tumors at days 7 ( n = 5) and 21 ( n = 6) after tumor inoculation were pulsed with OVA protein in complete IMDM medium for 2 hours, followed by their irradiation and coculture with OT-I cells at a ratio of 1:10 for 72 hours. OT-I cell proliferation was measured by thymidine incorporation ( n = 5 per group). ( B ) Quantification of the percentage (left) and number (normalized to tumor weight) (right) of the indicated populations among intratumoral cDC1s from B16-OVA tumors at the indicated timepoints, based upon TMRM and MG co-staining ( n = 10 per group). ( C ) Quantification of the geometric mean fluorescence intensities (gMFIs) of MHC-I, MHC-II, CD40, CD80 or CD86 on [TMRM/MG] hi cDC1s from B16-OVA at the indicated timepoints after tumor inoculation ( n = 9 at day 14; 10 at day 7 or 21). ( D ) Violin plots show activity score of cDC1 [TMRM/MG] hi UP signature (generated in-house from genes upregulated in [TMRM/MG] hi cDC1s compared to [TMRM/MG] lo cDC1s; see Methods) in cDC1s from human kidney cancer (left) ( GSE154763 ) or human non-small lung cancer (NSCLC; right) ( GSE139555 ) at different clinical [i.e., TNM (tumor, node, metastasis)] stages. Within each box, horizontal lines denote median values; boxes extend from the 25 th to the 75 th percentile of each group’s distribution of values. ( E and F ) ATAC-seq analysis of sort-purified cDC1s from B16-OVA tumors isolated on days 7 and 21 after tumor inoculation ( n = 4 per group). Transcription factor footprinting (E) and motif enrichment (F) analyses were performed by comparing day 21 versus day 7, and the transcription factors were ranked by their Z-scores (E) or odds ratios (F). The transcription factors with > 1,000 binding sites were selected for ranking (E). Blue dots and red dots indicate transcription factors predicted to have decreased and increased activity at day 21, respectively (E and F), and NRF1 was identified as the most downregulated transcription factor in the footprinting analysis (E). ( G ) GSEA enrichment plots showing decreased cDC1 OPA1-activated signature (generated in-house from the downregulated genes in OPA1-deficient versus WT cDC1s; see Methods) (left) and increased OPA1-suppressed signature (generated in-house from upregulated genes in OPA1-deficient versus WT cDC1s; see Methods) (right) in intratumoral cDC1s from B16-OVA tumors at day 21 versus day 7 after tumor inoculation. FDR, false discovery rate; NES, normalized enrichment score. ( H ) Splenic and intratumoral cDC1s were isolated from B16-OVA tumor-bearing mice at days 7 and 21 after tumor inoculation, followed by immunoblot analysis of OPA1 and NRF1. ACTB was used as loading control. Densitometric quantification of OPA1 or NRF1 was performed and normalized to ACTB expression. Numbers show the relative expression of OPA1 or NRF1 in indicated cDC1 population relative to splenic cDC1s from B16-OVA-tumor-bearing mice at day 7. ( I ) Confocal imaging analysis of TOM20 in intratumoral cDC1s derived from B16-OVA tumors at days 7 and 21 after tumor inoculation. Scale bars: 10 μm (left). Quantification of volume of TOM20 per mitochondrion (right) ( n ≥ 1,000 mitochondria per group). ( J to L) Schematic of cDC1 therapy model (J). WT mice were inoculated with 1 × 10 6 B16-OVA (K) or MC38 (L) tumor cells. Splenic total cDC1s, [TMRM/MG] hi cDC1s or [TMRM/MG] lo cDC1s were sorted from B16-Flt3L tumor-bearing mice (for DC expansion) and then were cultured with 100 μg/ml OVA protein and 20 μg/ml poly I:C (K) or MC38 cell lysate and 20 μg/ml poly I:C (L) in complete IMDM medium for 2 hours, followed by washing and subcutaneous transfer to tumor-bearing mice at day 5 after tumor inoculation. PBS was injected into non-transfer control mice ( n = 5 per group). Tumor growth was monitored. ( M and N ) Splenic [TMRM/MG] hi cDC1s or [TMRM/MG] lo cDC1s were sorted and pulsed with OVA protein and poly I:C as in (K). B16-OVA tumor-bearing mice received adoptive transfer of the indicated cDC1 subpopulations (or PBS) on day 6 after tumor inoculation. Anti-PD-L1 (aPD-L1; 200 mg) (or isotype control antibody) was intraperitoneally injected at days 7, 10 and 13 after tumor inoculation ( n = 5 per group). Tumor growth was monitored (M). Sixty days later, non-immunized (naïve) WT mice ( n = 4) and tumor-free mice ( n = 5) from the [TMRM/MG] hi cDC1s plus aPD-L1 treatment group were rechallenged with B16-OVA. Tumor cell growth was monitored (N). ( O and P ) Splenic [TMRM/MG] hi cDC1s or [TMRM/MG] lo cDC1s were sorted and pulsed with OVA protein and poly I:C as in (K) or MC38 cell lysate and poly I:C as in (L). WT mice with established B16-OVA (O) or MC38 (P) tumors received adoptive transfer of the indicated cDC1 subpopulations (or PBS) at day 6 after tumor inoculation. aCTLA-4 (200 mg) (O) or aPD-1 (200 mg) (P) (or isotype control antibody) was intraperitoneally injected at days 7, 10 and 13 after tumor inoculation ( n = 5 for each group). Tumor growth was monitored. Data are shown as mean ± s.e.m. in (A to C, I, and K to P). Two-tailed unpaired Student’s t -test in (A), two-way ANOVA in (B and K to P), one-way ANOVA in (C), two-tailed Wilcoxon rank sum test in (D), or Mann-Whitney test in (I). Data are representative of one (I), two (A to C, H, and M to P) or at least three (K and L). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. NS, not significant.

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: Membrane, Purification, Irradiation, Staining, Fluorescence, Activity Assay, Generated, Isolation, Footprinting, Binding Assay, Western Blot, Control, Expressing, Imaging, Derivative Assay, Cell Culture, Injection, Adoptive Transfer Assay, Two Tailed Test, MANN-WHITNEY