EMBO/soda-nel huggingface dataset:
TET2 promotes anti-tumor immunity by governing G-MDSCs and CD8+ T cell numbers (2020)
Figure 2

Figure 2. Tet2 knockout in mice impairs adaptive anti-tumor immune response.

A, B Immunohistochemistry analysis of CD8a, CD4 (A), PCNA, and Foxp3 (B) in tumors from WT and Tet2-/- (KO) mice subcutaneously injected with Hepa1-6 hepatoma cells. Corresponding bar charts display positive staining, quantified using Image Pro Plus 6.0 software (n=3). Pictures were captured at 400X magnification. Scale bar = 50 μm.

C Schematic illustration of the syngeneic tumor model in which Hepa1-6 cells were injected subcutaneously with or without anti-PD-1 treatment.

D Tumor growth curves in WT and KO mice treated with PBS or with anti-PD-1 Ab (n =3).

E Representatives images of day-19 tumors from WT and KO from panel D.

F Tumor weights from WT and KO mice at day 19.

G Flow cytometry analysis of splenic CD8+ T cells in WT and KO mice with or without anti-PD-1 treatment after tumor challenge.

H-K GSEA analysis showing the significant down-regulation of genes in GO biological processes: chemokine signaling pathway (H), T cell receptor signaling pathway (I), innate immune response (J) and cytokine receptor interaction (K). The x-axis represents that the mouse genes are ranked from the most up-regulated (left end) to the most down-regulated (right end) in comparison of syngeneic tumor from Hepa1-6 tumor-bearing KO to WT mice (n=4). The y-axis is an enrichment score (ES), which goes up (>0) and down (<0) representing the enrichment of up-regulated genes and down-regulated genes, respectively. NES is the normalized enrichment score. p-value was calculated by permutation.

Data information: In graphs, each symbol represents a mouse. Data were analyzed with two-tailed Student's t-test (A, B). *P < 0.05; **P < 0.01. Error bars denote ± SD. Data were analyzed with two-way ANOVA (D, F, G). *P < 0.05; **P < 0.01; ns, not significant.
TET2 promotes anti-tumor immunity by governing G-MDSCs and CD8+ T cell numbers (2020)
Figure 3

Figure 3. Tet2 deficiency results in G-MDSCs over expansion upon tumor challenge.

A Percentages of MDSCs (CD11b+Gr1+) gated from the CD45+ cell population in peripheral blood and spleen [n=11, tumor-free (-) WT and Tet2-/-（KO） mice; n=12, tumor-bearing (+) WT and KO mice, peripheral blood; n=10, tumor-free (-) WT and KO mice; n=13, tumor-bearing (+) WT and KO mice, spleen].

B Percentages of G-MDSCs (CD11b+Ly6G+) gated from the CD45+ cell population in peripheral blood and spleen [n=14, tumor-free (-) WT and KO mice; n=15, tumor-bearing (+) WT and KO mice, peripheral blood; n=15, tumor-free (-) WT and KO mice; n=15, tumor-bearing (+) WT and KO mice, spleen].

C Percentages of M-MDSCs (CD11b+Ly6C+) gated from the CD45+ cell population in peripheral blood and spleen [n=5, tumor-free (-) WT and KO mice; n=6, tumor-bearing (+) WT and KO mice, peripheral blood; n=6, tumor-free (-) WT and KO mice; n=6, tumor-bearing (+) WT and KO mice, spleen].

D Representative images of Ly6g and Ly6c immunohistochemistry in Hepa1-6 cell tumors from WT and KO mice. Corresponding bar charts display quantification of positive staining using Image Pro Plus 6.0 software (n=3). Pictures were captured at 400X magnification. Scale bar = 50 μm.

E Percentages of CD8+ T cells gated from the CD45+ cell population in peripheral blood and spleen [n=11, tumor-free (-) WT and KO mice; n=5, tumor-bearing (+) WT and Tet2-/- KO mice, peripheral blood; n=16, tumor-free (-) WT and KO mice; n=12, tumor-bearing (+) WT and KO mice, spleen].

F Percentages of CD4+ T cells gated from the CD45+ cell population in peripheral blood and spleen [n=8, tumor-free (-) WT and KO mice; n=5, tumor-bearing (+) WT and KO mice, peripheral blood; n=16, tumor-free (-) WT and KO mice; n=11, tumor-bearing (+) WT and KO mice, spleen].

Data information: Flow cytometric analysis of MDSCs and T cells in KO mice and littermate control mice (WT) with or without tumor challenge. In graphs, each symbol represents a mouse. ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001 using two-way ANOVA (A-C, E, F) and two-tailed Student's t-test (D). Error bars denote ± SD.
TET2 promotes anti-tumor immunity by governing G-MDSCs and CD8+ T cell numbers (2020)
Figure 4

Figure 4. Tet2 depletion expands the G-MDSCs population but does not affect the immunosuppressive function of G-MDSCs.

A Sorting efficiency of G-MDSCs (CD11b+Ly6G+) from spleens of tumor-bearing Tet2-/- （KO） and WT mice.

B GSEA analysis showing the significant upregulation of genes in GO biological processes: DNA replication (left), cell cycle G1-S phase transition (middle), and ribosome biogenesis (right). The x-axis represents that the mouse genes are ranked from the most upregulated (left end) to the most downregulated (right end) in comparison of tumor-bearing KO to WT mice from G-MDSCs of spleen (n=2). The y-axis is an enrichment score (ES), which goes up (>0) and down (<0) representing the enrichment of upregulated genes and downregulated genes, respectively. NES is the normalized enrichment score. p-value was calculated by permutation.

C G-MDSCs purified from spleen of tumor-bearing WT or Tet2-/- mice were co-cultured with CD8+ T cells at the indicated cellular ratios for T cell activation assays, quantified by CFSE-labeling and dye dilution for 3 days in vitro. Representative histograms show CFSE signals in the in vitro T cell activation assays.

D Quantification of CD8+ T cell proliferation in panel C. Each point represents a co-culture experiment (n=3). ns, not significant. Graphs show mean ± SEM and statistical analysis by Student's t-test.
TET2 promotes anti-tumor immunity by governing G-MDSCs and CD8+ T cell numbers (2020)
Figure 5

Figure 5. Excessive expansion of immunosuppressive G-MDSCs is the major cause of accelerated syngeneic tumor growth and decreased number of T lymphocytes in Tet2-/- mice.

A Schedule for anti-Ly6g antibody (Ab) treatment of Hepa1-6 tumor-bearing mice. Ab was intraperitoneally injected every 3 days as indicated. PBS was injected as a mock control.

B Representative flow cytometry plots of splenic G-MDSCs measured in tumor-free untreated Tet2-/- (KO) and WT mice and after anti-Ly6g or PBS injections into tumor-bearing mice.

C Tumor growth curves in WT and KO mice treated with either PBS or anti-mouse Ly6g (n=5).

D Day-18 tumor weights at the endpoint of (C) [n=11, PBS (-) injected tumor-bearing WT and KO mice; n=12, anti-Ly6g antibody (+) injected tumor-bearing WT and KO mice].

E Percentages of CD8+ T cells from the spleens of tumor-bearing mice with or without anti-mouse Ly6g treatment [n=17, PBS (-) injected tumor-bearing WT and KO mice; n=6, anti-Ly6g (+) antibody injected tumor-bearing WT and KO mice].

F Percentages of CD4+ T cells from the spleens of tumor-bearing mice with or without anti-mouse Ly6g treatment [n=16, PBS (-) injected tumor-bearing WT and KO mice; n=6, anti-Ly6g (+) antibody injected tumor-bearing WT and KO mice].

Data information: Flow cytometric analysis of T cells in Tet2-/- mice and littermate control mice (WT) with or without anti-Ly6g treatment after tumor challenge. In graphs, each symbol represents a mouse. ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001 using two-way ANOVA (C-F). Error bars denote ± SD.
TET2 promotes anti-tumor immunity by governing G-MDSCs and CD8+ T cell numbers (2020)
Figure 6

Figure 6. Increased IL-6 is responsible for the immunosuppressed phenotype of Tet2-/- mice.

A Gene set enrichment analysis of the IL-6/JAK/STAT signaling pathway in G-MDSCs from tumor-bearing Tet2-/- (KO) versus WT mice (n=2). p-value was calculated by permutation.

B ELISA analysis of IL-6, IFN-γ, and IL-2 levels in blood from WT and KO mice upon tumor challenge (n=4, tumor-bearing WT mice; n=3, tumor-bearing KO mice).

C Tumor growth curves in tumor-bearing WT and KO mice treated with PBS or anti-mouse IL-6 antibody (n=5).

D Day-18 tumor weights at the endpoint of (C) [n=10, PBS (-) injected tumor-bearing WT and KO mice; n=7, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice].

E, F Percentages of CD11b+ Ly6G+ G-MDSCs gated from total cells in (E) spleen and (F) peripheral blood from tumor-bearing mice treated with PBS or anti-mouse IL-6 antibody [n=5, PBS (-) injected tumor-bearing WT and KO mice; n=4, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice, spleen; n=4, PBS (-) injected tumor-bearing WT and KO mice; n=4, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice, peripheral blood].

G, H Percentages of CD8+ T cells gated from total cells in (G) spleen and (H) peripheral blood from tumor-bearing mice treated with PBS or anti-mouse IL-6 [n=5, PBS (-) injected tumor-bearing WT and KO mice; n=5, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice, spleen; n=5, PBS (-) injected tumor-bearing WT and KO mice; n=5, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice, peripheral blood].

I, J Percentages of CD4+ T cells gated from total cells in (I) spleen and (J) peripheral blood from tumor-bearing mice treated with PBS or anti-mouse IL-6 [n=5, PBS (-) injected tumor-bearing WT and KO mice; n=5, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice, spleen; n=4, PBS (-) injected tumor-bearing WT and KO mice; n=5, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice, peripheral blood].

Data information: Flow cytometric analysis of G-MDSCs and T cells in Tet2-/- mice and littermate control mice (WT) with or without anti-IL-6 treatment after tumor challenge. In graphs, each symbol represents a mouse. ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001 using two-way ANOVA (B-J). Error bars denote ± SD.

Expanded View Figure Legends
A key role for autophagy and the autophagy gene Atg16l1 in mouse and human intestinal Paneth cells (2008)
figf1

a, A gene-trap vector containing a splice acceptor followed by the &bgr;geo (&bgr;-galactosidase/neomycin resistance fusion cassette) exon disrupts the Atg16l1 gene locus by insertions within the intronic regions after exons 6 and 10 for the ATG16L1HM1 and ATG16L1HM2 lines, respectively. WT, wild type. b, MEFs were generated from day 12.5 embryos of Atg16l1 mutant mice, and western blot analysis was used to detect the &agr; and &bgr; isoforms of ATG16L1 from whole-cell lysates. c, d, Atg5-/- and ATG16L1HM MEFs were grown in the presence of the chemical inducer of autophagy rapamycin (Rap, 50 &mgr;g ml-1) and cyclohexamide (CHX, 5 &mgr;g ml-1) for 0, 4, 8 and 12 h. Cell lysates were analysed by western blot for loss of P62 expression (c). P62 levels were quantified by densitometry normalized to actin (n = 3; d). *P  0.04; **P  0.008; ***P = 0.0003. e, f, Western blot analysis of LC3 expression in MEFs grown in the presence of rapamycin (50 &mgr;g ml-1) or dimethylsulphoxide (DMSO) control for 4 h (e). LC3-II expression in the presence of rapamycin was quantified and normalized to actin (n = 6; f). g, Detection of ATG16L1, LC3 and P62 by western blot of ileal lysates from ATG16L1HM mice. ATG16L1 can be detected in both mutant lines on longer exposures. h, i, Quantification of ATG16L1 (h) and P62 (i) normalized to actin detected in ileal lysates (n = 3). P values were calculated using two-tailed Student’s t-test. Error bars, s.e.m.
A key role for autophagy and the autophagy gene Atg16l1 in mouse and human intestinal Paneth cells (2008)
figf2

a, b, Whole-mount images taken immediately above the ileal mucosal surface from WT (a) and ATG16L1HM (b) littermate mice stained with Helix pomatia lectin that labels mucus (green) and antisera directed against lysozyme (red). Images are representative of 3 WT and 3 ATG16L1HM mice. c, d, Ileal sections from WT (c) and ATG16L1HM (d) mice were stained with PAS/alcian blue to detect Paneth granules by light microscopy (dashed line denotes the crypt unit and arrowheads indicate Paneth cells). Images are representative of 7 WT, 7 ATG16L1HM1 and 5 ATG16L1HM2 mice; more than 100 crypts were analysed for each. e, f, Representative images of indirect immunofluorescence of sections stained for lysozyme (red) in WT (e) and ATG16L1HM (f) mouse ileal crypts. g, Paneth cells display one of four patterns of lysozyme expression (represented in red; white represents areas that exclude lysozyme): normal (D0), disordered (D1), depleted (D2) and diffuse (D3). h, i, Number of Paneth cells from ATG16L1HM (h) and Atg5flox/floxvillin-Cre (i) mice displaying each pattern of lysozyme expression (n = 6,660 cells from 5 ATG16L1HM mice and 5,634 cells from 5 WT mice; n = 1,756 cells from 2 Atg5flox/floxvillin-Cre mice and 1,649 from 2 Atg5flox/flox mice). Scale bars: a, b, 200 &mgr;m; c–f, 10 &mgr;m. *P  0.05, **P  0.01, ***P  0.001. P values were calculated using two-tailed Student’s t-test. Error bars, s.e.m.
A key role for autophagy and the autophagy gene Atg16l1 in mouse and human intestinal Paneth cells (2008)
figf3

a–d, Electron micrograph of littermate WT (a, c) and ATG16L1HM (b, d) Paneth cells (for a, b, dashed lines denote individual cells, asterisk indicates normal progenitor cell; for c, d, blue stars indicate normal mitochondria in WT Paneth cells and ATG16L1HM progenitor cells, red diamonds indicate degenerating mitochondria, the dashed box shows normal Golgi compartments, and yellow asterisks indicate granules; n = 3 mice per genotype). e, Mitochondrial integrity was quantified on the basis of the percentage of the visible mitochondrial section displaying intact cisternae (n = 49 WT and 71 ATG16L1HM cells). f, Enrichment of pathways associated with human orthologues mapped from genes differentially expressed in ATG16L1-deficient Paneth cells relative to WT cells, using the canonical pathway collection from MSigDB. The bar chart displays the negative log of the enrichment P values for each pathway using the hypergeometric distribution (see Methods). Pathways with only one gene assigned were excluded from the chart. † refers to the Wnt/Frizzled receptor-mediated cyclic GMP pathway obtained from the Signal Transduction Knowledge database (STKE, http://stke.sciencemag.org/cgi/cm/stkecm;CMP_12420). ‡ and ‡‡ denote similar pathways associated with &ggr;-hexachlorocyclohexane degradation as curated by KEGG and GenMAPP, respectively. g, Chart of selected genes for which mRNA is enriched in either ATG16L1-deficient Paneth cells (baseline is WT Paneth cells) or ATG16L1-deficient thymocytes (baseline is WT thymocytes). Only haemoglobin is in common in these two sets of analyses. NS, not significant. Scale bars: a, b, 10 &mgr;m; c, d, 500 nm.
A key role for autophagy and the autophagy gene Atg16l1 in mouse and human intestinal Paneth cells (2008)
figf4

a, b, Haematoxylin-and-eosin-stained sections of uninvolved areas from ileo–colic resections from patients with Crohn’s disease homozygous for the safe (a) or risk (b) allele of ATG16L1 (blue dashed line denotes crypt unit). c–f, Immunofluorescence images of Paneth cells from control patients (c, e) and patients with the risk allele (d, f) stained for lysozyme (red; c, d) and double-labelled additionally for leptin (green; e, f; yellow dashed line denotes the crypt unit). g, Aberrant lysozyme expression was quantified using the same criteria as that used for mouse sections in Fig. 3 (n = 6,829 cells for at risk patients and 8,182 for control, n = 5 patients for each genotype). Leptin-positive D3 cells were quantified in patient samples (n = 4 patients per genotype) homozygous for the risk allele (76 out of 322 D3 cells were positive from a total of 580 crypts examined) and homozygous for the safe allele (11 out of 93 D3 cells were positive from a total 749 crypts examined). Scale bars: a–f, 10 &mgr;m. *P  0.05, **P  0.01, ***P  0.001. P values were calculated using two-tailed Student’s t-test. Error bars, s.e.m.
Genetic control of pluripotency epigenome determines differentiation bias in embryonic stem cells (2021)
Figure 2

Figure 2: B6 EpiLCs are primed towards neuroectoderm lineage.

A-D. Example expression patterns for select genes identified by applying a general linear model (glm) including state, genotype, and interaction terms. Dots represent individual biological replicates (N = 3); lines highlight changes in mean values for replicates between each state (log2FC >1 and FDR < 0.05). A. State dependent expression is exemplified by Nanog which showed no difference between strains. B. Expression of Zfp277 exemplifies strain-dependence being consistently higher in B6. C & D. A significant genotype x state (GXS) interaction was identified for Nr5a2 (C, a marker for pluripotency) and Sox1 (D, a marker for neuronal differentiation).

E. Heatmap of transcript abundances for individual genes (rows) representing 12 expression modules detected by EBseqHMM filtered for genes with a significant GXS glm interaction. Genes with observed functional similarity within modules are highlighted. Nr5a2, Lifr, and other pluripotency genes shared the same expression path (m4) that decreases more significantly in B6, retained expression in D2, when ESCs are differentiated to EpiLCs. Sox1, Pax6 and other neurogenesis genes shared an expression path (m5) that is significantly up regulated in B6 in EpiLCs. Gstp2, Smarcd3 and other cell cycle regulation genes shared an expression path (m9) that is significantly up regulated in B6 ESCs.

F. Gene ontology (GO) enrichment of biological processes (FDR adjusted P value < 0.05) identified for 9 out of 12 expression modules (m1-9). Each column represents a GO term. Highlighted terms are indicated to the right. GO terms associated with proliferation (m4), neurogenesis (m5) and cell cycle regulation (m9) indicate genetic control of exit from pluripotency and early lineage priming.

G-I. Summarized differential module behavior between B6 and D2 (3 biological replicates) upon transition from ESCs to EpiLCs represented by PC1 of transcript abundance for all genes within each module. G. D2 retained proliferation in EpiLCs. H. Up regulation of neurogenesis in B6 EpiLCs. I. Increased regulation of cell cycle M phase in B6 ESCs.
Genetic control of pluripotency epigenome determines differentiation bias in embryonic stem cells (2021)
Figure 3

Figure 3. Embryoid bodies confirm genetic background influences differentiation propensity with B6 trajectory towards neuroectoderm.

A. UMAP embeddings of transcriptional profiles from spontaneously differentiated embryoid bodies (EBs). Each point represents a cell colored by genetic background and shaded by biological replicate (N = 3).

B. Similar to A indicating 13 major cell populations based on unsupervised clustering. Each cell is colored based on cluster.

C. Proportion of cells in a given cluster from B represented by either B6 or D2 genetic background.

D. Feature plot of expression gradients for indicated gene overlaid on UMAP from a. Three lineage markers that help to distinguish cellular identity are shown for five cell clusters.

E. Pseudocolor plots of FACS analysis on cells from EBs for both B6 and D2 strains. Cells were labeled with anti-EpCAM (top) or biological replicates with anti-SOX1 antibody (bottom). Gated population indicates percentages of EpCAM+ (Q3) or SOX1+ cells.

F. Bar chart showing percent positive cells gated for EpCAM+ population (left) or SOX1+ population (right). As predicted by scRNA-seq, more D2 EB cells express EpCAM compared to B6 and more B6 EB cells express SOX1 compared to D2 (N = 3, two-sided t-test, EpCAM p-value = 0.0012, and SOX1 p-value = 0.0048).
Genetic control of pluripotency epigenome determines differentiation bias in embryonic stem cells (2021)
Figure 4

Figure 4: trans-acting quantitative trait loci co-regulate chromatin accessibility and gene expression in ESCs and EpiLCs.

A. PCA of total transcript abundance in ESCs and EpiLCs from parental (3 biological replicates) and 33 distinct recombinant inbred BXD strains generated from crosses between B6 and D2. PC1 captures variation between cell state (57.6%) and PC3 captures transcript variation within cell states.

B. Scatterplot of genomic locations of individual chromatin accessibility (ca)QTL (x-axis, LOD > 5) versus location of ATAC peak being regulated (y-axis) for ESCs. Genetic effects that act locally (n = 3,973), i.e. in cis, fall on the diagonal line, while distal acting QTL (n = 13,767) lie off the diagonal. Several prominent distal QTL hotspots appear as vertical lines.

C. Similar to B, plotting expression (e)QTL position versus gene location (LOD > 5, n = 701 local-eQTL, n = 357 distal-eQTL).

D. Number of distal caQTL in 1 Mb windows versus genomic location mapped in ESCs (maroon) and EpiLCs (teal).

E. Similar to D comparing eQTL location and number between ESCs and EpiLCs.

F. Distribution of caQTL hotspot targets in relation to nearest transcription start site in ESCs as percentage of total targets.

G. Coverage profile for Gstp2 locus on Chr 19 showing H3K4me3, ATAC, and RNA read depth from merged replicates. Locations of Chr 7 trans caQTL targets are indicated with black bars and LOD scores listed below.

H. LOD score plot from QTL scan in ESCs for chromatin accessibility at the putative enhancer at Gstp2. Genetic variation at Chr 7 QTL distally regulates chromatin accessibility on Chr 19.

I, Phenotype by genotype plot of the Chr 7 caQTL from h. A B6 haplotype at Chr 7 is associated with increased chromatin accessibility on Chr 19 (horizontal line represents mean).

J&K. Similar to H&I showing QTL mapping and haplotype effect for expression of Gstp2 in ESCs (horizontal line represents mean).

L. Euler diagram showing overlap between genes in the neurogenesis module (m5 from Fig 2H) and Chr 12 trans eQTL (LOD >4) in EpiLCs (enrichment compared to all eQTL in neurogenesis module, Fisher's exact test - odds ratio (OR) = 7.78, p-value = 1.087e-06).