EMBO/soda-nel huggingface dataset:
The IL-6 signaling axis is a critical driver and therapeutic target in diffuse large B-cell lymphoma (2019)
Figure 2

Figure 2. Reconstitution of MISTRG mice with cord blood hematopoietic stem cells confers a growth advantage to xenotransplanted DLBCL cell lines in ectopic and orthotopic tumor models.

A-E MISTRG mice were reconstituted or not at birth with ~200.000 human CD34+ cord blood HSPCs, and subcutaneously transplanted with 10x106 U-2932 cells at six weeks of age. Tumor volumes were recorded over time, and along with tumor weights, at the study endpoint on day 27 post injection. The reconstitution efficiency (frequency of human among all human and mouse CD45+ leukocytes) is shown in A, and the average human leukocyte composition in the bone marrow of the mice in A is shown in B. Tumor volumes over time are presented in C along with tumor weights and volumes at the study endpoint in D, where each data point represents one tumor. The reconstitution level directly correlates with the tumor weight at the endpoint (E).

F-J MISTRG mice were reconstituted or not at birth with human CD34+ cord blood HSPCs, and intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age. The lymphoma burden at the study endpoint was quantified in the spleen (F) and bone marrow (G) by staining for hCD45 and by ZsGreen expression (as well as hCD19 and hCD20 for U-2932 and RC-K8, respectively), and in the case of RC-K8 cells, also over time by IVIS (H). The frequency of tissue involvement was assessed by ex vivo IVIS and macroscopic evaluation (I), and the occurrence of clinical symptoms (weight loss, hind leg paralysis) was recorded for each mouse (J). The x indicates that this mouse was not available for IVIS at the final timepoint because of premature death.

Data information: Data in A-E are pooled from three independent experiments. Data in F-J are pooled from two independent experiments per cell line. p-values were calculated using Mann-Whitney test and one-way ANOVA (in E). Horizontal lines indicate medians throughout. n is indicated in the figure panels throughout.
The IL-6 signaling axis is a critical driver and therapeutic target in diffuse large B-cell lymphoma (2019)
Figure 3

Figure 3. Genetic IL-6 humanization promotes growth of DLBCL cells that express the IL-6 receptor.

A-E MISTRG and MISTRG6 mice were intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age and monitored weekly by IVIS until the study endpoint (A). The lymphoma burden at the study endpoint (six weeks p.i. for RC-K8 and four weeks p.i. for U-2932) was quantified in the spleen (B) and bone marrow (C) by flow cytometric staining for hCD45 and by ZsGreen expression. The involvement of the indicated tissues as assessed by ex vivo IVIS is shown in D (solid bars, MISTRG; hatched bars, MISTRG6), and mice that have developed clinical symptoms (paralysis of hind legs) are plotted in E. In B,C and E, MISTRG is abbreviated as M and MISTRG6 as M6.

F Surface expression of the IL-6 receptor α-chain (CD126) as assessed by flow cytometry of U-2932 and RC-K8 cells. Data is representative of two stainings.

Data information: Data in A-E are from two independent experiments per cell line; ex vivo IVIS and clinical scoring of U-2932 (in D and E) was performed for only one experiment, and in vivo IVIS is shown for a subset of mice. Horizontal lines indicate medians throughout. p-values were calculated using the Mann-Whitney test. n is indicated in the figure panels throughout.
The IL-6 signaling axis is a critical driver and therapeutic target in diffuse large B-cell lymphoma (2019)
Figure 4

Figure 4. Human IL-6 and human immune system reconstitution both promote primary orthotopic DLBCL growth.

A-E 1x106 primary DLBCL cells obtained by passaging through a MISTRG6 mouse were intravenously injected into MISTRG or MISTRG6 recipients. The spleen weights (B, representative pictures in A) are shown along with representative H&E-stained spleen (MISTRG6) and lymph node (patient) sections in C and the lymphoma burden at the study endpoint eight weeks p.i., as quantified in the spleen and bone marrow in D and E. Representative flow cytometry plots are shown in D for bone marrow and spleen (MISTRG, MISTRG6). Lymphoma cells were positive for hCD19 and were lambda light chain-restricted (right panels of MISTRG6). M, MISTRG; M6, MISTRG6. Scale bar in A, 1 cm. Scale bar in C, 100 μm.

F MISTRG mice were reconstituted or not at birth with human CD34+ cord blood HSPC, and intravenously injected with 1x106 primary DLBCL cells at six weeks of age. The lymphoma burden at the study endpoint was quantified in the bone marrow of reconstituted and un-reconstituted animals by flow cytometric staining for the indicated surface markers.

G and H. Primary lymphoma cells harvested at the study endpoint express the IL-6R α chain (CD126, as assessed by flow cytometry, G) and the signaling chain gp130 (as assessed by immunohistochemistry, H) and are positive for phosphorylated STAT3 (G). Blue curves, hCD126 or pSTAT3; grey curves, isotype control antibody. Scale bar in H, 100 μm.

Data information: Data in A-E are pooled from two independent experiments and representative of three experiments. Data in F are from one experiment and representative of two. In B, E and F, each symbol represents one mouse and horizontal lines indicate medians. p-values were calculated using the Mann-Whitney test. n is indicated in the figure panels throughout.
The IL-6 signaling axis is a critical driver and therapeutic target in diffuse large B-cell lymphoma (2019)
Figure 5

Figure 5. The IL-6 receptor is expressed on a subset of DLBCL.

A-C The expression of the IL-6Rα (CD126) and signaling chains (gp130) was assessed by flow cytometry (A) and SYBR or Taqman qRT-PCR (B, C) respectively, on a panel of DLBCL cell lines. ABC- and GCB-DLBCL cell lines are color-coded in red and green; RC-K8 are depicted in grey, as their subtype is a matter of debate Kalaitzidis et al, 2002().

D and E. The expression of gp130 in 114 tumor biopsies of DLBCL patients was assessed by immunohistochemistry, and correlated with the ABC/non-GCB vs. GCB subtype, as assessed via Hans classification. Representative micrographs are shown in D, alongside the quantification of gp130+ cases among all samples, and among the two subtypes, in E. Scale bar, 100 μm.

Data information: The FACS plots in A are representative of at least two and up to four independent stainings per cell line. qRT-PCR results are presented as mean + SEM of two independent experiments. The p-value in E was calculated by Chi-square test. n.d., not detectable.
The IL-6 signaling axis is a critical driver and therapeutic target in diffuse large B-cell lymphoma (2019)
Figure 6

Figure 6. ABC-DLBCL cell lines depend on IL-6 for STAT3 activation.

A IL-6 secretion of the indicated cell lines (ABC-DLBCL in red, GCB-DLBCL in green) and primary patient material, as quantified by ELISA and normalized to cell number (shown per 1000 cells). n.d., not detectable.

B-D STAT3 phosphorylation as assessed by Western blotting (B, D) and flow cytometry (C) of the indicated ABC-DLBCL and GCB-DLBCL cell lines, with and without 1h of exposure to 50ng/ml hIL-6 (B,D). STAT3 expression levels are shown as Western blot control. Histograms in C represent STAT3 phosphorylation in the absence of exogenously added IL-6.

E STAT3 phosphorylation as assessed by Western blotting of CD126-proficient (WT) or -deficient (ko) RC-K8 cells, with and without 1h of exposure to 50ng/ml hIL-6.

F Kinetics of STAT3 phosphorylation as assessed by Western blotting, of the IL-6-expressing SOCS1wt and SOCS1mut cell lines OCI-Ly3 and SU-DHL-2, under 1, 6 and 24h exposure to an IL-6-neutralizing antibody (α-IL-6, anti-IL-6 neutralizing antibody).

G and H. STAT3 phosphorylation and gp130 expression of two representative cases (G) and summarized for the entire cohort of 114 patients (H), as assessed by immunohistochemistry. The specific pSTAT3 and gp130 signal is seen as brown (DAB staining), and counterstaining was performed using hematoxylin (blue nuclei). STAT3 phosphorylation is more common in biopsies with gp130 expression; the p-value was calculated by chi-square test. Scale bar, 100 μm.

Data information: Data in A are representative of two experiments and shown as mean + SEM. Data in B, D, E and F were reproduced in three independent experiments.
The IL-6 signaling axis is a critical driver and therapeutic target in diffuse large B-cell lymphoma (2019)
Figure 7

Figure 7. IL-6 signalling contributes to lymphoma development and germinal center B-cell proliferation in a genetically engineered mouse model

A and B Disease-specific mortality (A) and lymphoma burden as determined by lymph node weight (B), of cohorts of 9-12 mice each (n indicated in figure) of the indicated genotypes.

C-H Mice of the indicated genotypes were immunized once with sheep red blood cells and assessed 10 days later by flow cytometry of splenocytes for their frequencies of GC B-cells (C), centroblasts (D) and centrocytes (E). Spleen sections were further stained for Ki67 to reveal GCs (F), which were quantified both in number (G) and area (H). Arrows in F point to GCs; scale bar, 1000 μm.

Data information: Data in A and B are pooled from three (A) and two (B) different litters. Data in C-H are pooled from three independent studies. Statistical comparisons in B-H were performed either by one-way ANOVA (in the case of normal data distribution) or by non-parametric ANOVA (Kruskal Wallis test, in the case of non-normal data distribution) with Tukey's multiple comparisons correction. n is indicated in the figure panels throughout.
The IL-6 signaling axis is a critical driver and therapeutic target in diffuse large B-cell lymphoma (2019)
Figure 8

Figure 8. The IL-6 signalling complex is of diagnostic relevance and a therapeutic target in IL-6R+ DLBCL.

A-D Frequency of STAT3 phosphorylation (A), and overall as well as disease-specific survival probability (B) and of overall and disease-specific mortality of DLBCL patients stratified according to subtype and disease stage (C and D). Of all 814 evaluable patients, 367 were classified as GCB and 376 were classified as non-GCB/ABC-DLBCL according to the Hans algorithm. Phospho-STAT3 positivity was defined as expression in ≥17% of tumor cells and more common found in ABC-DLBCL tumors, and in more advanced (stage 3 and 4, relative to stage 1 and 2) disease (A). pSTAT3 positivity was a strong negative prognostic factor in the entire cohort (B) and associated with higher overall- as well as disease-specific mortality in both ABC- and GCB-DLBCL subtypes (C and D).

E-H Effect of IL-6R neutralization by twice-weekly injection of 250 μg/dose of tocilizumab or an isotype control antibody, starting two weeks post tumor cell injection, on (E and F) the spleen weights of MISTRG6 mice transplanted with 1x106 primary DLBCL cells (expanded by one serial passage through MISTRG6 mice), on the bone marrow lymphoma burden of OCI-Ly3-transplanted MISTRG mice (G) and on the splenic lymphoma burden of RC-K8-transplanted MISTRG6 mice (H), all assessed at around four (G and H) to eight (E) weeks post tumor cell injection. Representative microscopy images in F show STAT3 phosphorylation in spleen-colonizing primary cells exposed to either tocilizumab or isotype control antibody (in brown, counterstained in blue with hematoxylin), and an H&E-stained section from the same mouse. TCZ, tocilizumab. Scale bars, 100 μm.

Data information: Results in E, G and H are pooled from two (E and G) or three (H) independent studies each. Horizontal lines indicate medians. P-values were calculated by chi-square test (A,C,D), by log-rank test (B) or by Mann-Whitney test (E,G,H). n is indicated in the figure panels throughout.

Expanded View Figure Legends
Integrated analyses of single-cell atlases reveal age, gender, and smoking status associations with cell type-specific expression of mediators of SARS-CoV-2 viral entry and highlights inflammatory programs in putative target cells (2020)
Figure 1.

A cross-tissue survey of ACE2+TMPRSS2+ cells shows enrichment in cells at reported sites of disease transmission or pathogenesis. (a,b) Dual positive cells are more prevalent in epithelial organs and cells. (a) Proportion of ACE2+TMPRSS2+ cells (y axis) per dataset (dots) from 21 tissues and organs (rows). (b) Proportion of ACE2+TMPRSS2+ cells (y axis) within cell clusters (dots) annotated by broad cell-type categories (rows) within each of the top 7 enriched datasets (color legend, inset). (c,d) Significant co-expression of ACE2+TMPRSS2+ or ACE2+CTSL+ highlights cells from tissues implicated in transmission or pathogenesis. Significance of co-expression (dot size - log10(adjusted P-value), Methods; red border: FDR<0.1) of ACE2+TMPRSS2+ (c) or ACE2+CTSL+ (d) and effect size (dot color, color bar) for finely annotated cell classes (columns) from diverse tissues (rows). Only tissues and cells in at least one significant co-expression relationship are shown (Methods). (e-i) In situ validation of dual positive cells in the lung, airways, and submucosal gland. (e-g) PLISH and immunostaining in human adult lung alveoli for (e) ACE2 (white), TMPRSS2 (green) and CTSL (red), (f) ACE2 (white), TMPRSS2 (green) and HTII-280 (red), (g) ACE2 (red) and Pro-SFTPC (green). (h) PLISH and immunostaining in human large airway submucosal glands. ACE2 (red), ACTA2 (green) and DAPI (blue). (i) PLISH in human adult large airway. ACE2 (green) and DAPI (blue).
Integrated analyses of single-cell atlases reveal age, gender, and smoking status associations with cell type-specific expression of mediators of SARS-CoV-2 viral entry and highlights inflammatory programs in putative target cells (2020)
Figure 2.

Open chromatin at the ACE2 and TMPRSS2 loci in AT2, ciliated and secretory cells in the lung and airways (a,c) Single-cell ATAC-seq of lung samples from primary carina (1C) and subpleural parenchyme (RPL) (n=1 patient, k=3 samples, 3,366 cells from 1C, 8,340 cells from RPL). Uniform Manifold Approximation and Projection (UMAP) embedding of scATAC-seq profiles (dots) colored by (a, left) cell types, (a, right) cells with at least 1 fragment (indicating accessibility, open chromatin) mapping to the ACE2 gene locus (defined as −2kb upstream the Transcription Start Site to Transcription End Site), or by sample location (c). Grey shaded area: epithelial cell types. (b) Inferred gene activity of ACE2, TMPRSS2, CTSL across cell types. Log normalized mean "scATAC activity score" (quantified from accessibility, open chromatin) (dot color) and proportion of cells with active scores (dot size) for ACE2, TMPRSS2, and CTSL (columns) across different cell types (rows) from the primary carina (1C) and subpleural parenchyme (RPL). (d) Some AT2, ciliated and secretory cells have accessible chromatin at both ACE2 and TMPRSS2 loci. Proportion of cells (x axis) in each cell type (y axis) with accessible chromatin (at least 1 fragment) at both the ACE2 and TMPRSS2 loci (defined as −2kb upstream of the Transcription Start Site to Transcription End Site).
Integrated analyses of single-cell atlases reveal age, gender, and smoking status associations with cell type-specific expression of mediators of SARS-CoV-2 viral entry and highlights inflammatory programs in putative target cells (2020)
Figure 3.

ACE2, TMPRSS2, and CTSL expression in AT2 cells increases with age and smoking status, and in men (a) Samples in the aggregated lung and airway dataset partition to several classes by their cell composition. Percentage of cells (y axis) by level 2 cell annotations (Annotations with a preceding "1" indicate coarse annotations) across samples (x axis). The 282 samples are ordered by sample composition clusters. (b) Schematic of key lung and airway cell types highlighted in the study. (c) Statistical model. Model fitted to the data to assess sex, age, and smoking status associations with expression of the three genes. Yi,j denotes gene counts and nUMI denotes the total UMI counts per cell. (d) Age, sex, and smoking status associations with expression of ACE2 (blue), TMPRSS2 (orange), and CTSL (green) in epithelial cells. Effect size (x axis) of the association, in log fold change (sex, smoking status) or slope of log expression with age. Colored bars: associations with an FDR-corrected p-value<0.05, where pseudo-bulk analysis shows a consistent effect direction. Error bars: standard errors around coefficient estimates. (e) Distribution of ACE2 and TMPRSS2 expression across level 3 lung cell types. Red shading indicates the main cell types that express ACE2 and TMPRSS2. (f) Hold out analysis shows the robustness of associations to holding out a dataset. The values show the number of held-out datasets that result in loss of association between a given covariate (rows) and ACE2, TMPRSS2, or CTSL expression in a given cell type (columns). Robust trends are determined by significant effects that are robust to holding out any dataset (0 values). (g) Low expression in pediatric samples. Mean expression level (log CPM, y axis) of ACE2 (blue), TMPRSS2 (orange), and CTSL (green) across age bins (x axis) in AT2 (left) and ciliated (right) cells. Pediatric samples: 0-10 years. Samples from past or current smokers were removed from this plot to avoid smoking confounders. Error bars are omitted due to y-axis limitations. They are typically 10-fold the mean value (Supplementary Table 5). Multiciliated and AT2 cells are shown as these cell types are present in fetal data, and show significant age associations with ACE2 expression.
Integrated analyses of single-cell atlases reveal age, gender, and smoking status associations with cell type-specific expression of mediators of SARS-CoV-2 viral entry and highlights inflammatory programs in putative target cells (2020)
Figure 5:

Ace2, Tmprss2 and Ctsl2 expression in similar cell types and follows similar patterns with age and smoking. (a) Gradual increase in Ace2 expression by airway epithelial cell type with age. Mean expression (y axis) of Ace2 in different airway epithelial cells (x axis) of mice of three consecutive ages (color legend, upper right). Shown are replicate mice (dots), mean (bar), and error bars (standard error of the mean (SEM)). (b) Increase in proportion of Ace2+Ctsl+ goblet and club cells with age. Percent of Ace2+Ctsl+ cells (x axis) in different airway epithelial cell types (y axis) of mice of three consecutive ages (color legend, upper right). Shown are replicate mice (dots), mean (bar), and error bars (SEM). (c-j) Increase in Ace2 expression in secretory cells with smoking. Mice were daily exposed to cigarette smoke or filtered air as control for two months after which cells from whole lung suspensions were analyzed by scRNA-seq (Drop-Seq). (c,d) UMAP of scRNA-seq profiles (dots) colored by experimental group (c) or by Ace2+ cells and indicated double positive cells (d). Alveolar epithelial cells (AT1 and AT2) and airway epithelial secretory and ciliated cells are marked. (e) The relative frequency of Ace2+ cells is increased by smoking in airway secretory cells but not AT2 cells. Relative proportion (y axis) of Ace2+ (red) and Ace2- (grey) cells in smoking and control mice of different cell types (x axis). (f, g) Expression of Ace2 is increased in airway secretory cells, but not in AT2 cells. Distribution of Ace2 expression (y axis) in secretory (f) and AT2 (g) cells from control and smoking mice (x axis). (h-j) Re-analysis of published bulk mRNA-Seq101 of lungs exposed to different daily doses of cigarette smoke show increased expression of (h) Ace2, (i) Tmprss2, and (j) Ctsl after five months of chronic exposure. (k) Expression in placenta. UMAP plots showing expression of Ace2, Tmprss2 and Ctsl in single and double positive cells from embryonic days 9.5 to 18 (left) and embryonic day 14.5 (right) of mouse placenta development
Integrated analyses of single-cell atlases reveal age, gender, and smoking status associations with cell type-specific expression of mediators of SARS-CoV-2 viral entry and highlights inflammatory programs in putative target cells (2020)
Figure 6.

ACE2-protease co-expression and SARS-CoV-2 S-protein cleavage sites suggest a possible role for additional proteases in infection. (a) Multiple proteases are co-expressed with ACE2 in human lung scRNA-seq. Scatter plot of significance (y axis, -log10(adjusted P value)) and effect size (x axis) of co-expression of each protease gene (dot) with ACE within each indicated epithelial cell type (color). Dashed line: significance threshold. TMPRSS2 and PCSKs that significantly co-expressed with ACE2 are marked. (b) ACE2-protease co-expression with PCSKs, TMPRSS2 and CTSL across lung cell types. Significance (dot size, -log10(adjusted P value)) and effect size (color) for co-expression of ACE2 with selected proteases (columns) across cell types (rows). (c,d) Multiple proteases are expressed across lung cell types. (c) Distribution of expression (y axis) for ACE2, PCSKs and TMPRSS2 across lung cell types (x axis). White dot: median expression. (d) Proportion of cells (y axis) expressing ACE2, PCSK family or TMPRSS2 across lung cell types (x axis), ordered by compartment. (e) ACE2+PCSK+ dual positive cells across lung cell types. Fraction (y axis) of different ACE2+PCSK+ or ACE2+TMPRSS2+ dual positive cells across lung cell types (x axis). Dots: different individuals, line: median. (f) ACE2-protease co-expression analysis for the 20 most significant human proteases in AT2 cells. Significance (dot size, -log10(adjusted P value)) and effect size (color) for co-expression of ACE2 with different proteases (columns) across cell types (rows). (g) Additional protease expression in ACE2+TMPRSS2+ dual positive cells. Significance (y axis) and fold change (x axis) of differential expression for each human protease between ACE2+TMPRSS2+ dual positive vs dual negative cells within each indicated epithelial cell types (color). The top 20 most significantly differentially expressed proteases within AT2 cells and PCSKs across all epithelial cell types are highlighted.
GPR15-C10ORF99 functional pairing initiates colonic Treg homing in amniotes (2021)
Figure 3

Fig. 3 | GPR15-C10ORF99 functional pairing in amniotes.

(a-g) The cellular levels of Ca2+ ions after ligand (C10ORF99) stimulation of the indicated GPR15 receptors (human, mouse, Japanese quail, painted turtle, coelacanth, and Asian bony tongue GPR15). A plasmid encoding human NTSR1 and the parental pcDNA plasmid were transfected as positive and negative controls, respectively. The fold-change in calcium levels was calculated based on the fluorescence intensity (excitation and emission wavelengths: 490 and 520 nm, respectively). Calcium RLU (relative light unit) fold was calculated using no stimulation data as standard value.

(h) The cellular levels of Ca2+ ions after ligand (C10ORF99) stimulation of GPR15 in human, mouse, Japanese quail, painted turtle. YM.254890 inhibits Ca2+ ion levels in cells transfected with GPR15 receptors of vertebrates. The fold-change in calcium levels was calculated based on the fluorescence intensity (excitation and emission wavelengths: 490 and 520 nm, respectively).

(i, j) The cellular levels of Ca2+ ions after ligand (C10ORF99) stimulation of GPR15 in coelacanth, Asian bony tongue, and their mutants.

(k, l) The cellular levels of Ca2+ ions after ligand (C10ORF99) stimulating GPR15 in Japanese quail and its mutants. The fold-change in calcium levels was calculated based on the fluorescence intensity (excitation and emission wavelengths: 490 and 520 nm, respectively).
GPR15-C10ORF99 functional pairing initiates colonic Treg homing in amniotes (2021)
Figure 4

Fig. 4 | Functional conservation of GPR15-C10ORF99 pairing in amniotes.

(a-h) The migration of HEK293 cells expressing mGPR15, qGPR15, tGPR15, cGPR15, aGPR15, and their mutants induced by ligand C10ORF99.

(i-p) The migration of CD4+ cells from GPR15-knockout mice, engineered to express mGPR15, qGPR15, tGPR15, cGPR15, aGPR15, and their mutants induced by ligand C10ORF99.

Data information: All data shown are generated from at least three technical replicates. Error bars indicate SD (n=3). ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05 (t-test). Ad-NC represents adeno-virus-based gene-delivery vector as negative control.