Fig 1: (a) Soluble GARP (sGARP) cytokine suppression. CD4+ T cells were stimulated with 1 µg/mL anti-CD3 mAb, 1 µg/mL anti-CD28 mAb, and with or without 1 µg/mL sGARP for 24 h. INF-? production was measured by intracellular staining via flow cytometry. Dot plots show one representative result of 5 independent experiments. (b) T98G cells suppress T cell proliferation and cytokine production. CD4+ T cells were cultured together with or without (w/o) T98G cells in the ratio of 8:1 and stimulated, as described above. Additionally, either 10 µg/mL anti-GARP Ab or no Ab were added into the culture and CD4+ T cells were stimulated as described before. IFN-? production and proliferation (CFSE) were measured 4 days after stimulation by intracellular staining via flow cytometry. Dot plots show one representative result of 4 independent experiments. Data are displayed as mean values ± SEM, p-values relative to w/o ** p < 0.01. Dotted lines represent either unstained control (a + b IFN-?), CFSE stained cells before stimulation or percentages normalized to the untreated control (w/o).
Fig 2: GARP is shed from activated Treg and melanoma cellsGARP is found in Treg and melanoma supernatants. A. CD4+CD25+ Treg and CD4+CD25- Teff cells were stimulated with anti-CD3 and anti-CD28 mAb (each 1 µg/ml). Supernatants (SN) were removed at different time points (day 1 and day 4) and cells were lysed and analyzed by western blot (left) or ELISA (right) using anti-GARP mAb. Recombinant sGARP was used as control. Figure shows one representative result out of four independent experiments (n = 4, means ± SD, **P < .01, n.d. = not detectable). B. Melanoma cells (UKRV-Mel-15A) were cultured with or without PMA (50 ng/ml) for four hours. GARP expression was analyzed by flow cytometry. Bar diagram shows one representative experiment out of 4 (n = 4, means ± SD, *P < .05). C. Supernatants were removed from melanoma cells cultured with PMA or untreated (w/o) at different time points and analyzed by western blot using anti-GARP mAb. Recombinant sGARP was used as control. Shown is one representative result out of three independent experiments.
Fig 3: Flow cytometric and confocal analysis of GARP expression in patient-derived GB cell lines. (a) Confocal images show a strong GARP expression on the surface, intracellular (IC) and intranuclear (IN) in all tested patient-derived GB cell lines. The white bar corresponds to 20 µm (b) Flow cytometric analysis of the surface expression of GARP. All three cell lines showed an expression of GARP. Due to the nature of these cells, Nestin instead of DiO was stained. Means were normalized to the unstained control.
Fig 4: Glycoprotein A repetition predominant (GARP) immunohistochemistry in gliomas and astrocytomas. (a,d,e) Low-grade astrocytoma (WHO grade II) with more than 50% positive (pos.) labeled nuclei (magnification × 400). (b,f) GB with palisading necroses and more than 90% pos. stained tumor cells (magnification × 400). (c) Largely normal brain tissue in the neighborhood of a glioma with some labeled neurons (arrow) while others where unstained (arrowhead). Bar corresponds to 50 µm.
Fig 5: Analysis of the GARP localization in resting and stimulated regulatory T cells, melanoma cell line MaMel-19, and glioblastoma cell line T98G. The Treg were stimulated with 1 µg/mL anti-CD3 mAb, 1 µg/mL anti-CD28 mAb, and 10 IU/mL IL-2 for 48 h. (a) Cytoplasmatic and intranuclear localization of GARP shown in confocal images. The white bar corresponds to 20 µm (b) flow cytometric analysis GARP expression on the surface; surface and intracellular (IC); and surface, IC, and intranuclear (IN) of Treg, melanoma, and GB cell lines. Means were normalized to the unstained control.
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