EMBO/soda-nel huggingface dataset:
Helminthic dehydrogenase drives PGE2 and IL-10 production in monocytes to potentiate Treg induction (2022)
Figure 4

Figure 4. CLys and CSN mediate Treg induction from naïve CD4+T cells via PGE2 and IL-10 secretion from monocytes.

A Levels of bioactive LMs and IL-10 released from monocyte and non-monocyte fractions.

B Non-monocyte fractions were pre-incubated with IL-10R inhibitor and PGE2 receptor (EP2 and EP4) antagonists (or DMSO as a control) and incubated with CLys (left upper panel) or CSN (10µg/ml) (left lower panel) in presence or absence of monocytes and Treg induction was analysed after 72h. On the right panel, PGE2 receptors were first blocked on monocytes (2) or non-monocytes (3) or both (4) before addition of non-monocytes (2) or monocytes (3) and incubation with CSN for 72h and analysis of Treg induction compared to control condition (1).

C Treg induction by CLys and CSN in presence of monocyte/non-monocyte fractions and naïve CD4+CD45RA+CD45RO- T cells.

D Monocyte phenotyping in presence of CVF, CLys and CSN by FACS using CD68, CD80, CD163 and CD206 surface expression.

E Levels of LMs and PGE2 released by macrophages in presence of CVF, CLys and CSN Additionally, macrophages were lysed and gene expression of PTGS2, PTGES, ALOX5, ALOX15 and MaR1 quantified by RT-PCR.

F Treg induction following naïve CD4+CD45RA+CD45RO- T cells incubation with CLys-, CSN-monocyte conditioned media.

Data information: Heat maps in (A) and (D) show relative amounts of LMs detected by LC-MS/MS in supernatants. Data and graphs are representative of 6-7 biological replicates (n=6-7) for (A, B, C, D, F) and 4 biological replicates (n=4) for (E). Results are expressed as means ± SEM. Asterisks show significant statistical differences analyzed using Kruskal-Wallis one-way ANOVA followed by a Dunn's multiple comparison test. * p<0.05; ** p< 0.01.
Helminthic dehydrogenase drives PGE2 and IL-10 production in monocytes to potentiate Treg induction (2022)
Figure 5

Figure 5. Taenia solium GDH and IDH license Treg induction from naïve CD4+ T cells via PGE2 and IL-10 secretion.

A Induction of Treg following PBMC treatment with either native or proteinase K-treated CLys or CSN (10µg/ml) for 72h.

B Mass spectrometry analysis of CVF, CLys and CSN and identification of distinct and shared proteins. From the shared proteins in CLys and CSN, relative detection scores of GDH and IDH were presented.

C PBMCs were treated with GDH inhibitor bithionol (BT) (20µM) or IDH inhibitor IDH-305 (50nM) alone or in combination before incubation with CLys or CSN. Percentage of Treg was determined and PGE2 and IL-10 levels were evaluated.

D Treg induction from PBMC by CLys, CSN and TsGDH (5µg/ml) and levels of PGE2 and IL-10 in culture supernatants.

E Treg induction from monocyte-naïve CD4+-TsGDH culture and levels of PGE2 and IL-10 in monocyte-CD4+-TsGDH and monocyte-TsGDH culture supernatants.

F Treg induction from PBMC by CLys, CSN and rhGDH (10µg/ml).

Data information: Graphs are representative of 5-7 biological replicates (n=5-7) for (A-E) and 8 biological replicates (n=8) for (F). Results are expressed as means ± SEM. Asterisks show significant statistical differences analyzed using Mann-Whitney U test (A) or Kruskal-Wallis one-way ANOVA followed by a Dunn's multiple comparison test (C). * p<0.05; ** p< 0.01; *** p<0.001.
Zebrafish facial lymphatics develop through sequential addition of venous and non-venous progenitors (2019)
Figure 3

Figure 3. Spatiotemporal distribution of lymphatic progenitors in the cranial vessels

(A-H) Lateral images of the CCV fixed at 36 hpf (A) and 42 hpf (B), of the PHS at 36 hpf (C), 42 hpf (D) and 48 hpf (E) and of the VA-L at 36 hpf (F), 42 hpf (G) and 48 hpf (H) in lyve1b:EGFP fish that have been fluorescently immunostained with anti-PROX1 (green) and anti-GFP (red). PHS Prox1 staining shown alone for all time points (C'-E') with the FLS demarcated (dotted line) from the adjacent vessels, and the positions of the PHS-LPP and PHS-LPA indicated. Note the Prox1-postive (green), GFP-negative structure near to the VA-L is a prox1-expressing neuromast (NM).

Data information: CCV = common cardinal vein, FLS = facial lymphatic sprout, hpf = hours post fertilisation, NM = neuromast, PHS = primary head sinus, PHS-LPA = anterior primary head sinus lymphatic progenitor domain, PHS-LPP = posterior primary head sinus lymphatic progenitor domain, VA-L = ventral aorta lymphangioblast. Scale bar = 50 µm
Zebrafish facial lymphatics develop through sequential addition of venous and non-venous progenitors (2019)
Figure 6

Figure 6. The VA-L is not derived from a vein, instead it arises independently from a single angioblast cell

(A-B') Lateral (A) and ventrolateral (B, B') images of the cranial vessels in kdrl:nlsKaede;kdrl:EGFP embryos, with unconverted tissue shown in green (A-B) while photoconverted tissue is shown in red (B'). The early kdrl-positive VA-L was photoconverted in 34 hpf embryos (A), and followed through to 54 hpf (B, B'). B' is a higher magnification image of the box in B and is accompanied by a red channel only (traced photoconverted cells) image with the VA-L (dotted line) demarcated from the adjacent VA. The VA-L photoconverted cells are indicated by arrowheads.

(C) Still images from Movie EV5 of VA-L development in a kdrl:EGFP;kdrl:nlsmCherry compound transgenic from 31-35.5 hpf. This movie shows that the VA-L begins expressing kdrl just after 32 hpf, in a manner that is isolated and independent of any neighbouring kdrl-positive tissue or vessel. Putative kdrl-positive primitive myeloid cells (light blue asterisks) can also be seen migrating along the surface of the yolk in the immediate vicinity of the VA and VA-L.

(D) Still images from Movie EV6 depicting a 3D reconstruction of the 35.5 hpf timepoint from Movie EV5. A dorsal view that scrolls through the Y-stack of the VA-L is shown, which illustrates that the VA-L is closely associated with, but not connected to the VA.

(E-E') Still images from Movie EV7 of VA-L development in an etv2:EGFP (E), kdrl:nlsmCherry (E') compound transgenic from 28-35.2 hpf. This movie shows that the VA-L begins expressing etv2 (E) at 29 hpf and is independent of any neighbouring vessel. kdrl expression (red arrowheads) occurs 4 hours later in the VA-L (demarcated by the green dotted line) (E').

(F-G') Lateral images of the cranial vessels in etv2:Kaede embryos, with unconverted tissue shown in green (F-G) while photoconverted tissue is shown in red (G'). The earliest visible form of the VA-L was photoconverted at 29 hpf (F), then followed through to 54 hpf (G). G' is a higher magnification image of the box in G and is accompanied by a red channel only (traced photoconverted cells) image with the VA-L (dotted line) demarcated from the adjacent VA, and VA-L photoconverted cells indicated (arrowheads).

Data information: CCV = common cardinal vein, FLS = facial lymphatic sprout, hpf = hours post fertilisation, hpc = house post conversion, VA = ventral aorta, VA-L = ventral aorta lymphangioblast. Scale bar = 50 µm
Cellular responses to halofuginone reveal a vulnerability of the GCN2 branch of the integrated stress response (2022)
Figure 1

Figure 1. HF induces an atypical blunted ISR

A Representative immunoblots of the indicated proteins in lysates from HeLa cells treated with indicated concentrations of halofuginone (HF) for 5 h.

B Quantification of ATF4 and P-eIF2α from experiments as in (A). Data are mean ± SD (n = 3 biological replicates). *p ≤ 0.0414, **p ≤ 0.0019, ****p < 0.0001, as determined by one-way ANOVA with Dunnett's multiple comparison test.

C Similar to (A), but with HeLa cells treated with indicated concentrations of tunicamycin for 5 h.

D Similar to (A), but using lysates from HeLa cells treated with 62.5 nM and 312.5 nM HF for indicated times.

E Relative abundance of the indicated mRNAs detected by qPCR in lysates from HeLa cells treated with indicated concentrations of HF for 5 h. Data are mean ± SD (n = 3 biological replicates). *p ≤ 0.0388, **p ≤ 0.0046, ***p ≤ 0.0008, as determined by one-way ANOVA with Dunnett's multiple comparison test.

Data information: Representative results of at least three independent experiments are shown in each panel.
Cellular responses to halofuginone reveal a vulnerability of the GCN2 branch of the integrated stress response (2022)
Figure 2

Figure 2. GCN2 mediates eIF2α phosphorylation by HF

A Representative immunoblots of indicated proteins in lysates from mouse embryonic fibroblasts (MEFs) of indicated genotype after treatment with 12.5 nM or 200 nM HF for 5 h.

B Similar to (A), but using HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF for 5 h.

C Similar to (A), but with high HF concentrations.

Data information: Representative results of at least three independent experiments are shown in each panel.
Cellular responses to halofuginone reveal a vulnerability of the GCN2 branch of the integrated stress response (2022)
Figure 4

Figure 4. HF decreases R15B abundance independently of GCN2 and protein degradation

A Fold change of ATF4 and R15B after 5 h treatment with 12.5 or 312.5 nM HF relative to untreated cells. Data obtained from the quantitative proteomic analyses shown in Figure 3A.

B Representative immunoblots of indicated proteins in lysates from HeLa cells after 5 h treatment with indicated concentrations of HF.

C Similar to (B), but using HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF.

D Representative immunoblots of lysates from HeLa cells treated with indicated concentrations of HF for 5 h with or without the Nedd8-activating enzyme inhibitor MLN4924 (1 μM).

E. Representative immunoblots of lysates from HeLa cells treated with indicated concentrations of HF for 5 h with or without proteasome inhibitor MG-132 (10 μM).

Data information: Representative results of at least three independent experiments are shown in each panel.
Cellular responses to halofuginone reveal a vulnerability of the GCN2 branch of the integrated stress response (2022)
Figure 6

Figure 6. Translation attenuation in response to tRNA-synthetase inhibitors is independent on the ISR

A Newly synthesized proteins pulse-labeled with 35S-methionine for 10 min in HeLa cells pre-treated with indicated compounds for 2.5h, except (E) (Bottom) Coomassie-stained gel.

B Newly synthesized proteins in HeLa cells treated with 2.5 μg/ml Tm or 50 μg/ml cycloheximide (CHX).

C Newly synthesized proteins in 4KO or 4KO+GCN2 MEF cells treated with 12.5 or 200 nM HF.

D Newly synthesized proteins in eIF2αS/S and eIF2αA/A MEF cells treated with 12.5 or 200 nM HF.

E Newly synthesized proteins in eIF2αS/S and eIF2αA/A MEF cells treated with indicated concentrations of Tm for 2.5 or 5 h.

F Newly synthesized proteins in HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF.

G Newly synthesized proteins in HeLa cells treated with indicated concentrations of HF with or without proline supplementation (10 mM).

H Representative immunoblots of indicated proteins in lysates from HeLa cells after treatment with indicated concentrations of HF with or without proline supplementation (10 mM).

I Newly synthesized proteins in HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of histidinol (His) or borrelidin (Bor).

Data information: Quantification of experiments such as the ones shown in (C), (D) and (I) are presented in Figure EV3 and EV4.
Neutrophils suppress tumor infiltrating T cells in colon cancer via matrix metalloproteinase mediated activation of TGFβ (2019)
Figure 1

Figure 1. T cell depletion leads to increased colon tumor growth in mice.

A, Apcfl/fl-Cdx2CreERT2 mice were treated with Tamoxifen and, beginning the day following treatment, injected with either anti-CD4 and anti-CD8 neutralizing antibodies (αCD4/αCD8, blue dots) or IgG control (black dots) twice a week for six weeks.

B, FACS analysis of relative TCRβ+ T cell content in blood (left panel) and tumors (right panel) of mice at the end of treatments as indicated in A. αCD4/αCD8: n=10; IgG: n=6.

C, Tumor burden (left panel), tumor number (middle panel) and average tumor size (right panel) per mouse assessed during colon resection at the end of treatments. αCD4/αCD8: n=10; IgG: n=9.

Data information: In (B - C), statistical analysis was performed by unpaired two-tailed Student's t-tests. *: p<0.05; ****: p<0.0001. Exact p-values are provided in Appendix table S4.
Neutrophils suppress tumor infiltrating T cells in colon cancer via matrix metalloproteinase mediated activation of TGFβ (2019)
Figure 2

Figure 2. T cell exclusion in mouse colon tumors correlates with neutrophil and monocyte tumor infiltration.

Immune cell contexture in healthy colon and colon tumors of Apcfl/fl-Cdx2CreERT2 mice.

A, Relative TCRβ+ T cell content in colon (n=10) and tumor (n=22).

B, Anti-CD3 immunostaining on normal colon (left panel) and a representative colon tumor section derived from an Apcfl/fl-Cdx2CreERT2 mouse (right panel: higher magnification of area indicated in middle panel).

C, Relative CD11b+ myeloid cell content in colon (n=16) and tumors (n=16).

D, Anti-Gr1 immunostaining on normal colon (left panel) and a representative colon tumor section derived from an Apcfl/fl-Cdx2CreERT2 mouse (right panel: higher magnification of area indicated in middle panel).

E - G, Relative CD11b+ MHCII- Gr1hi neutrophil (E) and CD11b+ MHCII- Gr1lo monocyte (F) content in colon (n=13) and tumors (n=16). G, Correlation of tumor T cell numbers to sample-matched tumor-derived neutrophil (left panel) and monocyte (right panel) numbers (n=16).

H-J, Percentage of T cell subsets among total CD45+ hematopoietic cells in colon (n=10) and tumor (n=11). H, CD4+ TCRβ+ T cells. I, CD8+ TCRβ+ T cells. J, Foxp3+ CD4+ TCRβ+ regulatory T cells (Tregs).

K, Percentage of Tregs among total T cells (CD45+ TCRβ+) in normal colon (n=10) or colon tumors (n=11).

Data information: In (A), (C) and (E-K), relative content of individual cell types was determined in tissue-derived single cell suspension using cell type specific antibodies and FACS analysis. Each dot represents pooled tumors from one mouse. Statistical analysis performed by unpaired two-tailed Student's t-tests. *: p<0.05; ***: p<0.001; ****: p<0.0001. Exact p-values are provided in Appendix table S4.
Neutrophils suppress tumor infiltrating T cells in colon cancer via matrix metalloproteinase mediated activation of TGFβ (2019)
Figure 3

Figure 3. Tumor neutrophils suppress T cells in vitro and in vivo.

A, In vitro co-culture of activated T cells with increasing ratios of neutrophils, monocytes or macrophages. T cell proliferation index is numbers of proliferated T cells after three days of indicated co-culture condition relative to the number of proliferated T cells when cultured alone. CD4+ and CD8+ T cells were derived from lymph nodes of wild-type mice. Neutrophils, monocytes and macrophages were derived from colon tumors of Apcfl/fl-Cdx2CreERT2 mice. Each dot represents an individual neutrophil (n=4), monocyte (n=3) or macrophage (n=4) sample. Bars represent mean +/- SD.

B, Experimental setup. Eight weeks after Tamoxifen treatment of Apcfl/fl-Cdx2CreERT2 mice, animals were treated with anti-Gr1 antibody (αGr1, 3 times/week) plus CXCR2 inhibitor (CXCR2i, 5 times/week) or with IgG (3 times/week) plus DMSO control (5 times/week) for one to three weeks.

C, Tumor neutrophil (left panel) and monocyte (right panel) content after αGr1 + CXCR2i (n=7) or IgG + DMSO (n=10) treatments.

D, Average tumor volume per mouse at the end of αGr1 + CXCR2i (n=9) or IgG + DMSO (n=12) treatments as indicated in B.

E, Anti-CD3 immunostaining on representative tumor sections derived from mice after indicated treatments.

F, FACS analysis of CD45+ TCRβ+ cells, CD45+ TCRβ+ CD8+ cells and CD45+ TCRβ+ CD4+ Foxp3+ cells in extracted tumors after αGr1 + CXCR2i (n=8) or IgG + DMSO (n=7) treatments. G, FACS analysis of IFNγ (left panel) and IL17A (right panel) expression in tumor extracted T cells after αGr1 + CXCR2i (n=7) or IgG + DMSO (n=7) treatments.

Data information: In (A), statistical analysis was performed by using two-way ANOVA with Dunnett's multiple comparison tests. *: p<0.05; **: p<0.01. In (C - D) and (F - G), shown are combined data of two independent experiments. Each dot represents pooled tumors from one mouse. Statistical analysis was performed by unpaired two-tailed Student's t-tests. *: p<0.05; **: p<0.01. Exact p-values are provided in Appendix table S4.
Neutrophils suppress tumor infiltrating T cells in colon cancer via matrix metalloproteinase mediated activation of TGFβ (2019)
Figure 4

Figure 4. Tumor neutrophils suppress T cells in vitro via MMP9-mediated TGF-beta activation.

A, Measurement of cytokines present in cell culture supernatants of T cells co-cultured with either tumor-derived neutrophils (T + Ne; n=3) or with tumor-derived monocytes (T + Mo; n=3). Cytokines with a log2 fold change >0.6 or <-0.4 were considered as differentially present in supernatants. Shown is a heatmap of scaled cytokines levels (z-score) differentially present in T + Ne compared to T + Mo. Adjusted p-values were calculated by moderated t-test (limma) and Benjamini post test and were <0.05 only for MMP9 and RELMβ. Supernatants of T cells alone (T only; n=2) served as internal control. T cells cultured with monocytes did not have differential presence of cytokines compared to T cells alone.

B, Co-immunostaining for MMP9 (green) and S100A9 (red) on a representative tumor section derived from an Apcfl/fl-Cdx2CreERT2 mouse. Right panel shows a higher magnification of indicated area of the left panel.

C - D, mRNA expression levels of Mmp9 (C) or Tgfb1, Tgfb2 and Tgfb3 (D) in indicated FACS sorted cell types isolated from mouse colon tumors (n=4). Data are derived from RNA-sequencing and are displayed as reads per kilo base per million mapped reads (rpkm).

E - H, Neutrophil - T cell co-cultures treated with vehicle (T + Ne), 10µm Galunisertib (T + Ne + TGFBRi), 10µm of the MMP inhibitor GM6001 (T + Ne + MMPi) or with 10µm Galunisertib plus 10µm GM6001 (T + Ne + TGFBRi + MMPi). E, G and H, T cell proliferation after three days of indicated co-culture conditions. T cell proliferation index is numbers of proliferated T cells under indicated condition relative to the number of proliferated T cells when cultured alone. F, Protein levels of free active TGFβ1 in cell culture supernatants of indicated conditions determined by ELISA.

Data information: In (C - H) each dot represents a measurement on an individually derived sample. E: n=9. F: T only: n=8; T + Ne: n=8; T + Ne + MMPi: n=6. G: n=6. H: n=6. In (E - H), Statistical analysis was performed by paired two-tailed Student's t-tests. *: p<0.05; **: p<0.01; ***: p<0.001. Exact p-values are provided in Appendix table S4. F and H, Bars represent mean +/- SD.
Neutrophils suppress tumor infiltrating T cells in colon cancer via matrix metalloproteinase mediated activation of TGFβ (2019)
Figure 5

Figure 5. MMP induced TGFβ signaling promotes tumor formation.

A - C, Evaluation of TGFβ signaling activity in mouse colon tumors after four consecutive daily injections of hosts with 40mg/kg of the MMP2/9-inhibitor SB3-CT (MMPi). A and B, Co-immunostaining for pSMAD3 (green) and IGFBP7 (magenta) on representative tumor-containing colon sections derived from Apcfl/fl-Cdx2CreERT2 mice treated with vehicle (A) MMPi (B). C, Quantification of pSMAD3+ cells (left panel) and pSMAD3 staining intensity per pSMAD3+ cell (right panel) on at least five arbitrarily selected tumor areas per section of MMPi (n=6) or vehicle (n=6) treated mice.

D and E, effect of in vivo TGFBRi and MMPi treatment of mice for two weeks on colon tumor formation. D, Experimental setup. Apcfl/fl-Cdx2CreERT2 mice were treated with Tamoxifen and, one day post treatment, injected with either MMPi (n=10) or the TGFBRi SB431542 (n=10) at 40mg/kg for two weeks with five injections per week. Vehicle treated mice: n=11. Average tumor size per mouse was assessed during colon resection one week after the end of treatments (E).

F, Model of activation of latent TGFβ stored in extracellular matrix (ECM) by neutrophil-secreted MMP9, leading to suppression of effector T cells and promotion of Tregs.

Data information: In (C) and (E), statistical analysis was performed by unpaired two-tailed Student's t-tests. *: p<0.05; **: p<0.01. Exact p-values are provided in Appendix table S4.
Neutrophils suppress tumor infiltrating T cells in colon cancer via matrix metalloproteinase mediated activation of TGFβ (2019)
Figure 6

Figure 6. Neutrophils and TGFβ activity associate with T cell suppression in human colon cancer.

A - C, evaluation of S100A9+ neutrophil and CD8+ T cell infiltration in histologic sections of human CRC tumors. A, Anti-S100A9 immunostaining on a representative human CRC section. Arrowheads indicate neutrophil infiltration at the tumor boarder. B, Co-immunostaining for CD8 (brown) and S100A9 (gray-blue) in the same CRC sample as shown in A. C, Quantification of S100A9+ cells (upper panel) and CD8+ T cells in different areas on CRC tumor sections containing tumor border with adjacent benign mucosa as determined by a specialized pathologist: "Tumor center" was tissue within tumor >200μm from tumor border, "Tumor border" was tissue within tumor <100μm from tumor border and "Adjacent benign mucosa" was mucosa tissue outside tumor <100μm from tumor border. Per section, all identifiable tumor border and adjacent benign areas, as well as three arbitrary tumor center areas were scored. Sections were derived from 10 individual patients.

D and E, Gene expression scores were generated as described in material and methods. Boxes are lower and upper quartiles with median as solid lines; horizontal lines define minimum and maximum; dots define outliers. D, Neutrophil scores in tumor samples categorized according to CMS subtypes. E, Comparison of T cell gene expression scores in CMS4 tumors within two publicly available CRC gene expression datasets (median, lower and upper quartiles; horizontal lines define minimum and maximum; dots define outliers). Samples are categorized according to the medians (high = >median; low = <median) of neutrophil and TGFβ gene expression scores. Upper panel: TCGA dataset. Lower panel GSE39582 dataset.

F, Proliferation of activated blood T cells in vitro in co-culture with increasing ratios of CRC patient-derived autologous blood neutrophils. CD3+ T cells and CD66b+ neutrophils were isolated and cultured individually for each patient. Bar graphs display relative numbers of proliferated T cells after three days of co-culture. Each dot per condition represents a neutrophil sample from an individual patient (n=13). Bars represent mean + SD.

G, Proliferation of activated blood T cells in vitro in co-culture with autologous blood neutrophils. Cells were derived from both CRC patients (n=2) and healthy volunteers (n=5). T cells and neutrophils were cultured at a 1:1 ratio and treated with vehicle (T + Ne) or 10µm Galunisertib (T + Ne + TGFBRi). Each line represents a neutrophil sample from an individual healthy volunteer.

Data information: In (C) and (G), statistical analysis was performed by paired two-tailed Student's t-tests. *: p<0.05; **: p<0.01. In (D - E), p-values were calculated with Mann-Whitney test. *: p<1x10-3; **: p<1x10-6; ***: p<1x10-8; **** p<1x10-10. In (F), Statistical analysis was performed by using one-way ANOVA with Dunnett's multiple comparison tests. *: p<0.05; ***: p<0.001; ****: p<0.001. Exact p-values are provided in Appendix table S4.

Extended view figure legends