Fig 2: PSGL-1 positive virions can be captured by P-selectin and transferred to HIV-permissive target cells for infection. A Schematic depicting the experimental workfolw: virus preparations were added to MadCAM-1 and/or P-selectin coated wells to allow for virion capture. Post-incubation, wells were washed extensively to remove unbound virus and then were either assayed for the amount of captured virus using p24 detection (top workflow), or TZM-bl cells were overlayed onto each well for virus transfer, ad infectivity measurements via luminescence readout (bottom workflow). B Experimental results from viruses produced through transfection of HEK293T, C infection of T cell lines, and D PBMC IIIB viruses in plate-based virus capture (left column) and transfer assays (right column). Results displayed are the mean ± SD of two experiments with samples tested in duplicate wells for B and C. P values were determined using an unpaired t test (*P < 0.05; ***P < 0.0001). Results shown in D are representative of two independent experiments tested with duplicate wells

Fig 3: Virion-incorporated PSGL-1 retains the ability to bind selectin family receptors. A Virion capture assays were performed with immunomagnetic beads armed with 0.5 μg of recombinant selectins (P-, E-, L-selectin) or MAdCAM-1 with normalized inputs of NL4-3 virus produced through transfection of HEK293T cells with various amounts of PSGL-1 DNA (0 ng, 2.5 ng, 250 ng for negative, low, and high phenotypes respectively). B Viruses produced in T cell lines, and C PBMC (IIIB generated in two separate PBMC donors, D1 and D2) were added to armed beads at their undiluted titre. Bead-associated virus was lysed and HIV-p24 Gag was quantified using p24 AlphaLISA as an indicator of the amount of virus capture. Results show the mean ± SD of two independent experiments in which each condition was tested in duplicate. The results of unpaired t tests with Bonferroni correction are shown (**P < 0.01, ***P < 0.001)

Fig 4: GDF-15 interferes with LFA-1-dependent adhesion of human T cells.a–c µ-slides were coated with CXCL12a 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, a4ß7 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 CXCL12a and Mg2+ ± 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 CD3e 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.