Journal: Nature Communications

Article Title: Tumor-derived GDF-15 blocks LFA-1 dependent T cell recruitment and suppresses responses to anti-PD-1 treatment

doi: 10.1038/s41467-023-39817-3

Figure Lengend Snippet: a Effects of recombinant human (rh)GDF-15 (added for 10 min) on CXCL12α−mediated adhesion of whole blood-derived CD45 + cells to activated human lymphatic endothelial cells (huLEC) were analyzed. Adherence was calculated based on the number of CD45 + cells as enumerated by flow cytometry ( n = 7 experiments). In b , adhering leukocytes were further characterized by multicolor staining ( n = 10 experiments). c Stimulated T cells from 6 donors were treated or not with rhGDF-15 for 20 min before being run in µ-slides over a layer of activated huLEC. 10 predefined fields of view were video-imaged for 5 s and the number of T cells adhering under hydrodynamic flow conditions was counted. Different shadings indicate different T cell donors. For reference, adhesion to non-activated huLEC is shown for one donor. d Enumeration of T cells adhering to human umbilical vein endothelial cells (HUVEC). 5 predefined fields of view per sample were analyzed in a representative experiment. In e , CXCL9 and CXCL10 were used to induce adhesion of untreated or GDF-15-treated CD8 + T cells from 3 different donors on stimulated huLEC. In f , stimulated CD8 + T cells from 3 different donors were treated with rhGDF-15 and anti-GFRAL or isotype control antibodies for 20 min before being run in µ-slides over a layer of activated huLEC. In g – j , phase-contrast microscopy in chamber slides to assess effects of rhGDF-15 on T cell adhesion to activated HUVEC. An EC 50 value for rhGDF-15-mediated adhesion inhibition on pan T cells from 3 different donors was determined in g (logICF=logIC50 + (1/HillSlope)*log(F/(100-F)). h – j Using pan T cells from 9 different donors, effects of rhGDF-15 on T cell adhesion ( h ), transmigration ( i ) and recruitment ( j ) were analyzed. Statistical analyses were performed by one-way ANOVA in a , d , f , by two-sided paired Student´s t -tests in b , e , h , i , j , by mixed-effects analysis in c . To correct for multiple comparisons, Tukey´s post hoc test was applied in a , c , d , Bonferroni´s method in b . In c , d , g , mean values with SEM, in e , f , median values are indicated as horizontal lines. Source data are provided as a Source Data file.

Article Snippet: Fibronectin was aspirated, followed by a wash with huLEC or HUVEC medium (InSCREENeX, Braunschweig, Germany). huLEC (InSCREENeX) or HUVECs (Lonza, Basel, Switzerland or freshly isolated) were detached, washed and resuspended at 1×10 6 cells/ml.

Techniques: Recombinant, Derivative Assay, Flow Cytometry, Staining, Control, Microscopy, Inhibition, Transmigration Assay

Journal: Nature Communications

Article Title: Tumor-derived GDF-15 blocks LFA-1 dependent T cell recruitment and suppresses responses to anti-PD-1 treatment

doi: 10.1038/s41467-023-39817-3

Figure Lengend Snippet: a – c µ-slides were coated with CXCL12α and vehicle or ICAM-1-Fc ( a–c ), MAdCAM-1-Fc ( b ), or VCAM-1-Fc ( c ). Stained primary human T cells were stimulated with anti-CD3/CD28 before GDF-15, or vehicle, or antibodies against adhesion molecules LFA-1, α4β7 integrin, or VCAM-1 were added for 30 min. T cells were perfused for 6 min over the coated µ-slides. Adhesion was recorded by live microscopy and analyzed using CellProfiler software. In d , e , CD4 + and CD8 + T cells were pre-treated for 20 min with GDF-15, or blocking anti-LFA-1 antibody TS1/18, or both, and run over activated HUVEC as in ( 1e – i ). f , g Binding of conformation-specific anti-active LFA-1 antibody mAb24 ( f ) or ICAM-Fc ( g ) to CD8 + T cells was analyzed. Whole blood from healthy volunteers was maintained at 37 °C and treated or not with GDF-15 10 min prior to LFA-1 activation. Fluorescence-conjugated antibodies and complexed soluble ICAM-1-Fc were added for another 10 min. Cells were fixed and analyzed on an Attune Nxt flow cytometer. Mean fluorescent intensity (MFI) values were normalized to control conditions by z-transformation. h , i , j Human PBMC were stimulated for 30 min with CXCL12α and Mg 2+ ± rhGDF-15. Cells were stained with the conformation-specific Alexa Fluor 647-labeled anti-LFA-1 antibody mAb24 ( h ) or hICAM-1-Fc-AF647 ( i ). The number of active LFA-1 molecules per single CD3 + T cell was quantified by direct stochastic optical reconstruction microscopy. Representative single cell images are shown in h , i . Data obtained with mAb24 across three different donors are summarized in j . k , l T cells were added to ICAM-1- and E-Selectin-coated Protein G beads, in the absence or presence of GDF-15. After lysis, Talin phosphorylation was assessed by Western blotting, with CD3ε as loading control. A representative blot is shown in k . Protein quantification data from 7 different samples normalized to vehicle (human serum albumin) control are displayed in l . Statistics were calculated by Kruskal–Wallis with Dunn´s post hoc test ( a ), by one-way ANOVA with Tukey´s correction for multiple comparisons ( b–e ), and by two-sided paired t -tests ( f , g , j , l ). Horizontal bars indicate mean ( a , f , g ) or median ( d , e , j , l ) values. Source data are provided as Source Data file.

Article Snippet: Fibronectin was aspirated, followed by a wash with huLEC or HUVEC medium (InSCREENeX, Braunschweig, Germany). huLEC (InSCREENeX) or HUVECs (Lonza, Basel, Switzerland or freshly isolated) were detached, washed and resuspended at 1×10 6 cells/ml.

Techniques: Staining, Microscopy, Software, Blocking Assay, Binding Assay, Activation Assay, Fluorescence, Flow Cytometry, Control, Transformation Assay, Labeling, Lysis, Phospho-proteomics, Western Blot

Journal: Advanced Healthcare Materials

Article Title: Design of Recombinant Spider Silk Proteins for Cell Type Specific Binding

doi: 10.1002/adhm.202202660

Figure Lengend Snippet: Cell type, tissue origin, and reasons for the selection of the cells. Abbreviations: HUVECs: human umbilical vein endothelial cells, hiPSC‐CMs: human induced pluripotent stem cell derived cardiomyocytes

Article Snippet: HUVEC from the human umbilical cord (INS‐Cl‐1002, InSCREENeX GmbH, Germany) were cultured in huVEC basal medium (INS‐ME‐1011) supplemented with 5.7 % v/v supplements (INS‐ME‐1011BS, both InSCREENeX GmbH, Germany) and 0.1 % v/v gentamycin sulfate in gelatine‐coated cell culture flasks. hiPSC derived cardiomyocytes were provided by Tilman Esser and Prof. Felix B. Engel (Nephropathology, University Hospital Erlangen, Germany) and cultured as published previously.

Techniques: Selection, Derivative Assay

Journal: Advanced Healthcare Materials

Article Title: Design of Recombinant Spider Silk Proteins for Cell Type Specific Binding

doi: 10.1002/adhm.202202660

Figure Lengend Snippet: Cell adhesion on films made of eADF4(C16) (cytophobic), eADF4(C16)‐RGD (cytophilic), and eADF4(C16)‐KGD (cell selective). Cell adhesion and spreading of rat B50 neuronal cells, mouse Balb 3T3 embryonal fibroblasts, human BJ fibroblasts, mouse C2C12 myoblasts, human HaCaT keratinocytes, human induced pluripotent stem cell derived cardiomyocytes (hiPSC‐CM), human HeLa cervix carcinoma cells, human umbilical vein endothelial cells (HUVEC), human MG63 bone fibroblasts, mouse‐rat NG108 neuronal hybrid cells, and rat RN22 Schwann cells on different spider silk films were recorded after 4 h of incubation using fluorescence microscopy. Therefore, cell nuclei and F‐actin cytoskeleton of fixed cells were stained using DAPI (blue) and Phalloidin (red), respectively. White arrows point towards round cellular aggregates showing small cell body sizes and preferring cell–cell‐contacts, but no/little cell–matrix interactions. White asterisks highlight clearly spread cells exhibiting enhanced cell body sizes. The yellow box marks the selectivity of C2C12 myoblasts on the eADF4(C16)‐KGD variant. Scale bars: 100 µm.

Article Snippet: HUVEC from the human umbilical cord (INS‐Cl‐1002, InSCREENeX GmbH, Germany) were cultured in huVEC basal medium (INS‐ME‐1011) supplemented with 5.7 % v/v supplements (INS‐ME‐1011BS, both InSCREENeX GmbH, Germany) and 0.1 % v/v gentamycin sulfate in gelatine‐coated cell culture flasks. hiPSC derived cardiomyocytes were provided by Tilman Esser and Prof. Felix B. Engel (Nephropathology, University Hospital Erlangen, Germany) and cultured as published previously.

Techniques: Derivative Assay, Incubation, Fluorescence, Microscopy, Staining, Variant Assay