Journal: eLife

Article Title: Tumor stiffening reversion through collagen crosslinking inhibition improves T cell migration and anti-PD-1 treatment

doi: 10.7554/eLife.58688

Figure Lengend Snippet: Migration of activated PBT plated onto fresh tumor slices was analyzed in EGI-1 and MMTV-PyMT tumor model, whilst resident tumor-infiltrating T lymphocytes were analyzed in mPDAC and KPC tumor model. Illustrative images of T cell migration tracks in EGI-1, MMTV-PyMT, mPDAC, and KPC tumor models. Tumor stroma (fibronectin) in red, tumor cells (EpCAM in EGI-1, MMTV-PyMT, and KPC tumor models, CD44 in mPDAC tumor models), in blue and T cells (CD8 in mPDAC and KPC, Calcein in MMTV-PyMT, and EGI-1 tumor models) in green. Tracks are color-coded to illustrate track displacement. Scale bar = 100 µm. T cell migration speed, T cell displacement, and trajectory straightness in all tumor models. ***p - value>0.001, p-value>0.05, Student’s t-test. Results are shown as mean ± SD. Figure 5—source data 1. Source data file for .

Article Snippet: To evaluate T cell migration in the EGI-1 model, activated human CD8+ T cells were first labeled with CellTrace Calcein red-orange dye (ThermoFischer).

Techniques: Migration

Journal: eLife

Article Title: Tumor stiffening reversion through collagen crosslinking inhibition improves T cell migration and anti-PD-1 treatment

doi: 10.7554/eLife.58688

Figure Lengend Snippet:

Article Snippet: To evaluate T cell migration in the EGI-1 model, activated human CD8+ T cells were first labeled with CellTrace Calcein red-orange dye (ThermoFischer).

Techniques: Derivative Assay, Recombinant, Cell Isolation, Activation Assay, Software

Journal: Tissue Engineering. Part A

Article Title: Engineered Liver Tissue Culture in an In Vitro Tubular Perfusion System

doi: 10.1089/ten.tea.2020.0213

Figure Lengend Snippet: Preparation of Vascular Channel for TPS. (A) Three-dimensional model of the porous central vascular channel designed in SOLIDWORKS (Dassault Systèmes) and the final 3D-printed construct. (B) Schematic of cell seeding approach for HUVECs (left), and the confocal microscopy outcome indicating a highly confluent monolayer of HUVECs pre-labeled by CellTracker Orange adhered to the outer and inner wall of the channel without blocking the micro-pores aligned on the construct. 3D, three-dimensional; HUVEC, human umbilical vascular endothelial cell; TPS, tubular perfusion system.

Article Snippet: Before the seeding, HUVECs were pre-labeled with an orange fluorescent dye by incubation with Celltracker™ Orange CMRA Dye (Thermofisher) in EGM for 30 min.

Techniques: Construct, Confocal Microscopy, Labeling, Blocking Assay

Journal: Nature Communications

Article Title: Dermal IRF4+ dendritic cells and monocytes license CD4+ T helper cells to distinct cytokine profiles

doi: 10.1038/s41467-020-19463-9

Figure Lengend Snippet: IRF4 WT → or IRF4 ∆CD11c → C57BL/6 bone marrow chimeras were immunized i.d. with Ms , Nb , Ca , or PBS as a control. LN cell suspensions were examined by flow cytometry on day 2 ( a , b ) or day 5 ( c – k ) after immunization. All conditions were compared within each experiment. a , b Cellular composition ( a ) and Ag median fluorescence intensity (MFI) ( b ) of LN Ag+ populations 2 days after immunization with CellTracker Orange-labeled Ms , Nb, or Ca . Graphs show mean ± SEM for groups of n = 9 ( Ms and Nb IRF4 WT ) or 8 ( Ca IRF4 WT and all IRF4 ∆CD11c ) female chimeras from two independent experiments. Statistical significance was assessed using a two-sided Student’s t test for each cell population. NS: not significant, ** p < 0.01, **** p < 0.0001. Exact values are shown for 0.05 > p > 0.01. c – k CD4+ T cell responses as assessed by flow cytometry. Intracellular cytokine staining was after in vitro restimulation, transcription factor (TF) expression was assessed without in vitro restimulation. c Numbers of CD4+ T cells per LN and ( d ) frequencies of CD44+ cells in the indicated CD4+ T cell populations. Bar graphs show mean values ± SEM, each symbol corresponds to one mouse. e Contour plots showing IFNγ staining of CD4+ T cells from Ms -immunized mice. Data are concatenated from 4 mice. f Frequencies of total cytokine+ and Tbet+ CD4+ T cells, or ( g ) IFNγ hi (red gates in ( e )) and Tbet hi CD4+ T cells, in Ms -immunized mice. h , j Contour plots showing IL-4 and IL-17A staining of CD4+ T cells from Nb - or Ca- immunized mice, respectively. Data are concatenated from 4 mice. i , k Frequencies of cytokine+ and TF+ CD4+ T cells in Nb- or Ca -immunized mice. All graphs show mean ± SEM for groups of n = 6 (PBS IRF4 WT and PBS IRF4 ∆CD11c ) n = 7 ( Ca IRF4 WT and Ms IRF4 ∆CD11c ) or n = 8 ( Ms, Nb IRF4 WT and Nb, Ca IRF4 ∆CD11c ) female chimeras over two independent experiments; each symbol in ( c , d ) corresponds to one mouse. Statistical significance was assessed using Two-Way ANOVA with Sidak’s post-test. NS: not significant; ##, ** p < 0.01; ###, *** p < 0.001; ####, **** p < 0.0001. Hash symbols refer to comparisons between PBS and immunized chimeras of the same genotype. Asterisks refer to comparisons between similarly immunized IRF4 WT or IRF4 ∆CD11c chimeras. Source data are provided as a Source Data file.

Article Snippet: For the pathogen mixing experiments in Fig. , and experiments in IRF4 ΔCD11c mice which express GFP in CD11c+ cells, pathogens were labeled with CellTracker™ Orange CMTMR (CTO, Invitrogen™, ThermoFisher Scientific, MA) or Cell Proliferation Dye eFluor670 (eBioscience, ThermoFisher Scientific, MA) according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Fluorescence, Labeling, Staining, In Vitro, Expressing

Journal: Nature Communications

Article Title: Dermal IRF4+ dendritic cells and monocytes license CD4+ T helper cells to distinct cytokine profiles

doi: 10.1038/s41467-020-19463-9

Figure Lengend Snippet: Mice of the indicated mouse strains were immunized i.d. with fluorescently labeled pathogens. LN cell suspensions were examined on day 2 after immunization unless otherwise indicated. a Expression of Il12b and Cxcl9 transcripts and cell surface CD11c protein on total monocytes. RT-qPCR was on monocytes sorted from the pooled LN of five C57BL/6 female mice; each symbol refers to one biological replicate, n = 3. CD11c median fluorescence intensity (MFI) data are from groups of n = 6 (PBS) or four (immunized) female mice examined in one of five independent experiments that gave similar results; each symbol refers to one mouse. Bar graphs show mean ± SD. b Expression of CD11c on Ag− and Ag+ monocytes following Ms immunization. Contour plots show concatenated data from three mice, the line graph shows means ± SD for groups of n = 3 ( Ms d1 and Ms d3) or 4 ( Ms d2) female mice examined in one of two independent experiments that gave similar results. c Monocyte numbers in mice treated with neutralizing anti-IFNγ antibodies or isotype control. The bar graph shows mean ± SEM for groups of n = 10 (PBS) or 11 ( Ms -immunized) female mice examined over two independent experiments; each symbol refers to one mouse. d Numbers of monocytes and IFNγ+ NK cells in mice treated with neutralizing anti-IL-12p40 + anti-IL-18 antibodies or isotype controls and immunized with Ms . Bar graphs show mean ± SEM for groups of n = 12 (monocytes) or 11 (NK cells) female mice examined over two independent experiments; each symbol refers to one mouse. e Expression of IFNγ-induced markers on monocytes from mice treated with neutralizing anti-IL-12p40 + anti-IL-18 antibodies or isotype controls and immunized with Ms . CD11c and Ly6A/E bar graphs show mean ± SEM for groups of n = 6 female mice from one of two independent experiments that gave similar results. CXCL9 bar graphs show mean ± SEM for groups of n = 11 (PBS) and 15 ( Ms -immunized) female mice examined over 2 independent experiments. Symbols refers to individual mice. f Expression of Il12b -YFP in Ag− and Ag+ migDC2 and monocytes 2 and 3 days after immunization with CellTracker Orange-labeled Ms . Contour plots show concatenated data from 6 mice. g Frequencies of Il12b -YFP+ Ag− and Ag+ migDC2 and monocytes 2 and 3 days after Ms immunization. Bar graphs show mean ± SEM for groups of n = 6 male mice, each symbol refers to one mouse. Data are from one of three independent experiments that gave similar results. h Number of NK cells and frequency of IFNγ+ or CD11b+ NK cells in the indicated mouse strains 1 or 2 days after Ms immunization. Line graphs show mean ± SEM for groups of n = 10 (5 male + 5 female WT), 6 (3 male + 3 female BATF3-KO PBS), 8 (5 male + 3 female BATF3-KO Ms and CCR2-KO PBS), or 9 (CCR2-KO Ms ) mice over two independent experiments, and 8 (5 male + 3 female IRF4 WT or IRF4 ∆CD11c PBS) or 10 (5 male + 5 female IRF4 WT or IRF4 ∆CD11c Ms ) mice over two independent experiments. i Expression of Tbet and CXCR5 in CD4+ T cells from C57BL/6 (WT) or CCR2-KO mice 5 days after Ms immunization. Flow data are concatenated from five mice. Bar graphs show mean numbers±SEM for groups of n = 10 (PBS and Ms WT) or 9 ( Ms KO) female mice tested over two independent experiments. Each symbol refers to one mouse. Statistical significance was assessed using One-Way ANOVA with Holm–Sidak’s post-test ( a , c , d , e , i frequencies), two-Way ANOVA with Sidak’s post-test ( b , g ), mixed-effects ANOVA with Sidak’s post-test ( h ), or unpaired two-tailed Student’s t test ( i ratio). NS: not significant, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Exact p -values are shown for 0.05 > p > 0.01. Source data are provided as a Source Data file.

Article Snippet: For the pathogen mixing experiments in Fig. , and experiments in IRF4 ΔCD11c mice which express GFP in CD11c+ cells, pathogens were labeled with CellTracker™ Orange CMTMR (CTO, Invitrogen™, ThermoFisher Scientific, MA) or Cell Proliferation Dye eFluor670 (eBioscience, ThermoFisher Scientific, MA) according to the manufacturer’s instructions.

Techniques: Labeling, Expressing, Quantitative RT-PCR, Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Cas9-Mediated Knockout of Ndrg2 Enhances the Regenerative Potential of Dendritic Cells for Wound Healing

doi: 10.1101/2022.03.14.484360

Figure Lengend Snippet: a , Single-cell RNA sequencing (scRNA-seq) of vitamin D3 (VD3) stimulated and untreated bone marrow-derived dendritic cells (BM-DCs) (10X Genomics Chromium), cells colored by experimental group. b , cells colored by Seurat clusters. Premat = premature. c , Vegfa and Ndrg2 expression projected onto UMAP embedding in untreated and VD3 stimulated cells. d , Subset of cluster 8 showing Ndrg2+ progenitor cells, colored by experimental group. e , Expression of Ndrg2 (red) and Cd34, Itgae , and Flt3 (blue) projected onto UMAP embedding of cluster 8 subset. Right column shows merged expression of Ndrg2 and co-expressed genes. f , Flow cytometry of untreated and VD3 stimulated BM-DCs, gated on CD34, CD103, and CD135. g , Schematic of co-culture experiments using BM-DCs and human umbilical vein endothelial cells (HUVECs). h , Endothelial tube formation after co-culture of endothelial cells together with untreated DCs or VD3 and lipopolysaccharide (LPS) stimulated DCs. DCs were stained with calcein red, ECs were stained with calcein AM (green). Cell nuclei were stained with Hoechst (blue). Bottom row shows binary images created using the Angiogenesis Analyzer. i , Analysis of HUVEC branch number, branch length (pixels), junction number and total mesh area (pixels), (n = 3 biological replicates, one-way ANOVA: *P < 0.05, **P < 0.01, ***P < 0.001). j , Luminex multiplex ELISA showing protein expression of 33 differentially expressed proteins in cell culture media of untreated DCs and of DCs stimulated with either VD3, LPS or VD3 and LPS (n = 3 biological replicates, one-way ANOVA: **P < 0.01, ****P < 0.0001).

Article Snippet: For co-culture assays, DCs were stained using CellTrace Calcein Red-Orange (ThermoFisher) and added to the EC cultures at a 1:1 ratio.

Techniques: RNA Sequencing Assay, Derivative Assay, Expressing, Flow Cytometry, Co-Culture Assay, Staining, Luminex, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: bioRxiv

Article Title: Cas9-Mediated Knockout of Ndrg2 Enhances the Regenerative Potential of Dendritic Cells for Wound Healing

doi: 10.1101/2022.03.14.484360

Figure Lengend Snippet: a , UMAP embedding of single-cell RNA sequencing (scRNA-seq) data from Ndrg2-KO dendritic cells (DCs), vitamin D3 (VD3) stimulated DCs, and untreated cells. RNA velocity stream vectors computed with scVelo are projected onto the embedding. Cells colored by experimental group (23,871 cells). b , Cells colored by Seurat cluster. Cluster 9 represents Ndrg2+ progenitor cells. c , UMAP embedding split by experimental condition, and cells colored by cell density. In each plot, cells of the indicated condition are colored in yellow/violet and cells of the other two conditions are colored in grey. e , Violin plots of DC maturation markers. f , UMAP embedding split by experimental condition. Ndrg2+ progenitor cells (cluster 9) colored in red. g , Violin plots of wound healing related markers. h , Heatmap of the top 10 differentially expressed genes per cluster, sorted by average log fold-change and ordered by experimental condition. i , Over-representation analysis (ORA) of the indicated gene sets (GO-BP) projected on the UMAP embedding. j , Ndrg2-KO cells seeded on collagen-pullulan hydrogels stained with calcein AM. 3D reconstruction of stacked confocal microscopy images.

Article Snippet: For co-culture assays, DCs were stained using CellTrace Calcein Red-Orange (ThermoFisher) and added to the EC cultures at a 1:1 ratio.

Techniques: RNA Sequencing Assay, Staining, Confocal Microscopy