Fig. 7

Hypoxic GBM-derived EVs disrupt T cell attraction and reactivation capacity of macrophages. A-D Mouse BMDMs were unpolarized (M0), M1 polarized (M1), or M2 polarized (M2) for 7 days in the absence (contr.) or presence of hypoxic GBM-derived EVs (1% EVs) or normoxic GBM-derived EVs (21% EVs). A mRNA expression of Cxcl9, Cxcl10 and Il12b in BMDMs. mRNA levels were measured by RT-qPCR and expression was normalized to housekeeping genes (Gapdh and Eef1a1). B Macrophages were harvested, washed, then incubated for a further 24 h. Supernatants were collected and CXCL10 secretion was measured by a flow cytometry bead-based assay. C Macrophages were harvested, washed, pulsed with OVA peptide then added to OT-II CD4⁺ cells. IFN-γ secretion was measured after 24 h by ELISA. D (Left panel) Macrophages were harvested, washed, then added to the lower chambers of Transwell plates for 3 h. CD4⁺ OT-II T cells were added to the upper chamber and migration measured after 6 h. Negative migration control: CD4⁺ T cells were pretreated with pertussis toxin (PTX). Positive migration control: CXCL10 was added to the BMDMs in the lower chamber (CXCL10). E (Left, center and right panel) Human MDMs (M0) from three donors were harvested, washed, then added to the lower chambers of Transwell plates for 3 h. (Left and center) CD3⁺ T cells from the corresponding buffy coat donor were added to the upper chamber and the migration was measured after 6 h. Negative migration control: CD3⁺ T cells were pretreated with pertussis toxin (PTX). Positive migration control: CXCL10 was added to the human macrophages in the lower chamber. (Right panel) Identical experiment as in left and center panels with the use of CD3⁺ T cells from a different donor. Values are expressed as mean ± SD of three biologic replicates, and comparisons were made using an unpaired t test. *p < 0.05, **p < 0.005, ***P < 0.001, ****P < 0.0001