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