Journal: Science Advances

Article Title: Distinct thymic pDC populations promote tumor immune tolerance through complementary mechanisms

doi: 10.1126/sciadv.adx9864

Figure Lengend Snippet: ( A ) Flow cytometry analysis of DN thymocyte subsets in Ccr9 WT and Ccr9 KO mice treated with PBS or inoculated with MC38 tumors for 21 days ( n = 3 to 5 per group). DN thymocytes were gated on the basis of CD25 and CD117 expression into DN1 to DN4 stages. ( B ) Absolute numbers of DN1 to DN4 thymocytes in PBS-treated (P) or MC38 tumor-bearing (T) Ccr9 WT and Ccr9 KO mice. ( C ) Mean fluorescence intensity (MFI) of Notch1 expression in DN1 to DN4 thymocytes from PBS-treated or tumor-bearing mice. ( D ) Relative Dll4 mRNA expression in the thymus from PBS-treated or MC38 tumor-bearing mice at day 14, measured by qPCR ( n = 4 to 6 per group). ( E ) In vitro differentiation assay using sorted DN1, DN2, and DN3 thymocytes cultured with Tst4/N-DLL1 cells in the presence of 10% normal medium (NM) or tumor-conditioned medium (TM) for 7 days. BST2 + B220 + pDCs were analyzed after gating on CD11c + cells ( n = 3 per group). ( F ) Absolute numbers of BST2 + B220 + pDCs generated from DN1 to DN3 thymocytes under the conditions described in (E). ( G ) IFN-α production by thymic pDCs (left) and total CD11c + cells (right) isolated from PBS-treated (P) or MC38 tumor-bearing (T) mice at day 14 following 12-hour stimulation with ODN1585 CpG (Stim) or without stimulation (Unstim) ( n = 4 to 5 per group). ND, not detected. ( H and I ) Flow cytometry analysis of Qa2 expression in CD4SP (H) and CD8SP (I) thymocytes from Ccr9 WT and Ccr9 KO mice treated with PBS (P) or inoculated with MC38 tumors (T) for 14 days. Frequencies of Qa2 + cells are shown ( n = 4 to 7 per group). Data are expressed as means ± SEM. * P < 0.05; ** P < 0.01; n.s., not significant. Unpaired Student’s t test.

Article Snippet: The cells were incubated for 12 hours in the presence or absence of ODN1585 CpG (5 μM; InvivoGen).

Techniques: Flow Cytometry, Expressing, Fluorescence, In Vitro, Differentiation Assay, Cell Culture, Generated, Isolation

Journal: Life Science Alliance

Article Title: Histone methyltransferase DOT1L differentially affects the development of dendritic cell subsets

doi: 10.26508/lsa.202603624

Figure Lengend Snippet: (A) Cre-ER T2 Dot1l wt/wt and Cre-ER T2 Dot1l fl/fl mice were injected three times with tamoxifen. Subsequently, BM was cultured with SCF and FLT3L. Seven days after the start of the culture, DC and precursor subset composition was evaluated. (B) Comparison of myeloid and lymphoid precursor percentages of Live Lineage neg AF neg cells in Dot1l- WT (Cre-ER T2 Dot1l wt/wt ) and Dot1l -KO (Cre-ER T2 Dot1l fl/fl ) conditions. CMPs, MDPs, and CDPs were gated from MHC class II neg CD11c neg CD135 pos cells and defined as CD117 hi CD115 neg CMPs, CD117 int CD115 hi MDPs, and CD117 neg CD115 hi CDPs. CLPs were defined as MHC class II neg CD11c neg CD135 neg CD127 pos . Pre-pDCs were gated as CD117 neg CD135 pos CD11c pos Siglec-H pos Ly-6D pos , pDCs as B220 pos Siglec-H pos BST2 pos , and cDC1s and cDC2s as B220 neg MHC class II hi CD11c hi Sirpα neg/pos . (C) On day 7 after the start of the culture, BM cells were stimulated with class A CpGs (ODN1585) overnight. IFNα levels were determined using ELISA. (D) After class A CpG stimulation, cells were stained for maturation markers. Conditions are shown with and without the addition of CpGs. The individual dots represent individual mice with a total of three independent experiments performed. Error bars indicate the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: 3 × 10 6 BM cells or splenocytes were seeded in a round-bottom 96-well plate with medium (RPMI-1640 [Gibco] with 10% heat-inactivated FCS [Biowest], 50 U/ml penicillin [Lonza], 50 mg/ml streptomycin [Lonza], and 50 μM 2-mercaptoethanol) containing 1 μM ODN1585 (InvivoGen).

Techniques: Injection, Cell Culture, Comparison, Enzyme-linked Immunosorbent Assay, Staining

Journal: Life Science Alliance

Article Title: Histone methyltransferase DOT1L differentially affects the development of dendritic cell subsets

doi: 10.26508/lsa.202603624

Figure Lengend Snippet: (A) Schematic overview of the setup of the 7-d BM culture. Total BM of WT C57BL/6 mice was cultured with SCF and FLT3L to facilitate the development of DCs. DOT1L inhibitor SGC-0946 (1 μM) was added at days 0, 3, and 5 to ensure constant inhibition of DOT1L during DC development. DMSO (0.1%) was used as a vehicle control. (B) Comparison of myeloid and lymphoid precursor percentages of Live Lineage neg AF neg cells in cultures with and without DOT1L inhibitor. CMPs, MDPs, and CDPs were gated from MHC class II neg CD11c neg CD135 pos cells and defined as CD117 hi CD115 neg CMPs, CD117 int CD115 hi MDPs, and CD117 neg CD115 hi CDPs. CLPs were defined as MHC class II neg CD11c neg CD135 neg CD127 pos . Pre-pDCs were gated as CD117 neg CD135 pos CD11c pos Siglec-H pos Ly-6D pos , pDCs as B220 pos Siglec-H pos BST2 pos , and cDC1s and cDC2s as B220 neg MHC class II hi CD11c hi Sirpα neg/pos . (C) On day 7, BM cells were stimulated with class A CpGs (ODN1585) overnight. IFNα levels were determined in the supernatant using ELISA, and maturation markers were evaluated by flow cytometry. Conditions are shown with and without the addition of CpGs. The individual dots represent the average of technical replicates in one independent experiment, with a total of three independent experiments performed. Error bars indicate the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: 3 × 10 6 BM cells or splenocytes were seeded in a round-bottom 96-well plate with medium (RPMI-1640 [Gibco] with 10% heat-inactivated FCS [Biowest], 50 U/ml penicillin [Lonza], 50 mg/ml streptomycin [Lonza], and 50 μM 2-mercaptoethanol) containing 1 μM ODN1585 (InvivoGen).

Techniques: Cell Culture, Inhibition, Control, Comparison, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Life Science Alliance

Article Title: Histone methyltransferase DOT1L differentially affects the development of dendritic cell subsets

doi: 10.26508/lsa.202603624

Figure Lengend Snippet: (A) Schematic overview of the experimental setup. Cre-ER T2 Dot1l wt/wt and Cre-ER T2 Dot1l fl/fl mice were injected with tamoxifen on three subsequent days to induce KO of Dot1l . On day 12, BM was harvested and analyzed. (B) Comparison of myeloid and lymphoid precursor percentages of Live Lineage neg AF neg cells in Dot1l -WT (Cre-ER T2 Dot1l wt/wt ) and Dot1l -KO (Cre-ER T2 Dot1l fl/fl ) conditions. CMPs, MDPs, and CDPs were gated from MHC class II neg CD11c neg CD135 pos cells and defined as CD117 hi CD115 neg CMPs, CD117 int CD115 hi MDPs, and CD117 neg CD115 hi CDPs. CLPs were defined as MHC class II neg CD11c neg CD135 neg CD127 pos . Pre-pDCs were gated as CD117 neg CD135 pos CD11c pos Siglec-H pos Ly-6D pos , pDCs as B220 pos Siglec-H pos BST2 pos , and cDC2s as B220 neg MHC class II hi CD11c hi Sirpα pos . (C) Comparisons of specific markers on pDCs and DC2s that were differentially expressed between Dot1l -WT and Dot1l -KO conditions based on the MFI of (N = 10) biological replicates. (D) On day 12, BM cells were stimulated with class A CpGs (ODN1585). IFNα levels were determined in the supernatant using ELISA. In addition, cells were stained intracellularly for IFNαβ. After overnight stimulation, cells were also stained for maturation markers. Conditions are shown with and without the addition of CpGs. The individual dots represent individual mice with a total of two independent experiments performed. (E) Pie charts highlighting the number of up- and down-regulated genes in Dot1l-KO DC populations compared with Dot1l-WT as determined by RNAseq (based on Log 2 FC < −0.5 and >0.5 for the leftmost plot and <−1.0 and >1.0 for all others). (F) GO enrichment of genes that were down-regulated in KO pDCs (Log 2 FC < −1.0). (G) Heatmap of selected transcription factors showing the H3K79me2 signal near the transcriptional start site (TSS; N = 2, green gradient) and expression differences between Dot1l KO and WT (N-1, blue-red gradient) in the sorted DC subsets. (H) GO enrichment of genes that were up-regulated in expression in all sorted subsets (Log 2 FC > 0.5). For (F, H), pathways were ranked by fold enrichment and visualized using ShinyGO . (I) Heatmap correlating the H3K79me2 TSS signal to expression differences for genes from pathways enriched in all KO subsets (as visualized in (H)). Error bars indicate the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: 3 × 10 6 BM cells or splenocytes were seeded in a round-bottom 96-well plate with medium (RPMI-1640 [Gibco] with 10% heat-inactivated FCS [Biowest], 50 U/ml penicillin [Lonza], 50 mg/ml streptomycin [Lonza], and 50 μM 2-mercaptoethanol) containing 1 μM ODN1585 (InvivoGen).

Techniques: Injection, Comparison, Enzyme-linked Immunosorbent Assay, Staining, RNA sequencing, Expressing

Journal: Life Science Alliance

Article Title: Histone methyltransferase DOT1L differentially affects the development of dendritic cell subsets

doi: 10.26508/lsa.202603624

Figure Lengend Snippet: (A) Cre-ER T2 Dot1l wt/wt and Cre-ER T2 Dot1l fl/fl mice were injected with tamoxifen on three subsequent days to induce KO of Dot1l . On day 12, organs were harvested, and several (functional) experiments were performed on the splenocytes and BM cells. The region surrounding exon 2 of Dot1L was amplified, and the frequency of Dot1l ∆ alleles was quantified as described previously (BM, bone marrow). (B) Histograms of H3K79me2 in representative Dot1l WT and KO samples derived from either spleen (left) or BM (right). For the KO samples, cells overlapping with the WT peak were classified as H3K79me2 high, and the remainder as H3K79me2 low. (C) Quantification of the H3K79me2 low fractions from S5B (N = 10 for spleen, N = 2 (pooled replicates) for BM). (D) Splenic DC subset percentages of Live Lineage neg AF neg cells in Dot1l WT (Cre-ER T2 Dot1l wt/wt ) and Dot1l -KO (Cre-ER T2 Dot1l fl/fl ) conditions. pDCs were gated as BST2 pos Siglec-H pos , cDC2s as CD11c pos MHC class II pos Sirpα pos , and cDC1s as CD11c pos MHC class II pos XCR1 pos . (E) Mean fluorescence intensity (MFI) of proliferation marker Ki67 on several cell subsets in the BM (left) and spleen (right) in Dot1l WT versus KO conditions on day 12 after the start of tamoxifen injections. The individual dots represent individual mice with a total of two independent experiments performed. (F) Quantification of specific markers on pDCs and cDCs that were differentially expressed between Dot1l WT and Dot1l -KO conditions based on the MFI of (N = 10) biological replicates. (G) On day 12, splenocytes were stimulated with class A CpGs (ODN1585). IFNα levels were determined in the supernatant using ELISA. In addition, cells were stained intracellularly for IFNαβ. After overnight stimulation, cells were also stained for maturation markers. Conditions are shown with and without the addition of CpGs. (H) Principal component analysis of pooled and sorted DC subsets (N = 1) from both WT and Dot1l-KO mice. (I) Pie chart (left) highlighting the number of down- and up-regulated genes in all KO populations (based on Log 2 FC < −0.5 and >0.5, respectively); (right) GO enrichment of genes that were down-regulated in expression (Log 2 < −0.5) in all sorted subsets, ranked by fold enrichment and visualized using ShinyGO. (J) Representative H3K79me2 ChIPseq tracks for Ciita and Fbxo11 exported from IGV. Data scales were standardized to visualize quantitative differences both between genes and between DC subsets. (K) Heatmap of selected epigenetic modifiers showing the H3K79me2 signal near the TSS (N = 2, green gradient) and expression differences between Dot1l KO and WT (N = 1, blue-red gradient) in the sorted DC subsets. Error bars indicate the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Error bars indicate the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: 3 × 10 6 BM cells or splenocytes were seeded in a round-bottom 96-well plate with medium (RPMI-1640 [Gibco] with 10% heat-inactivated FCS [Biowest], 50 U/ml penicillin [Lonza], 50 mg/ml streptomycin [Lonza], and 50 μM 2-mercaptoethanol) containing 1 μM ODN1585 (InvivoGen).

Techniques: Injection, Functional Assay, Amplification, Derivative Assay, Fluorescence, Marker, Enzyme-linked Immunosorbent Assay, Staining, Expressing