Fig. 5

ARG1-EVs are internalized by murine bone marrow-derived DCs (BMDCs) and block DCs-primed T-cell proliferation. a Representative image of EVs endocytosed by BMDCs from confocal microscopy. BMDCs were incubated with 50 µg of PKH-67-stained ARG1-EVs isolated from the supernatants of ID8-ARG1-V5 murine ovarian carcinoma cell line for 4 h in 37 or 4 °C (inhibited internalization, negative control), washed and fixed. Green—PKH67-stained EVs, blue—DAPI nuclear stain. b Western blotting for V5-tag labeled ARG1 in DCs lysates. BMDCs were incubated with 50 µg of ARG1-EVs isolated from supernatants of ID8-ARG1-V5 murine OvCa cell line for 4 h in 37 °C, washed and lysed. ID8-ARG1-V5 (ID8-ARG1) cells lysate was used as a positive control for V5-tagged Arg1 detection. Beta-actin served as equal protein loading control. c Representative proliferation histograms of αCD3/αCD28-stimulated CD4⁺ and CD8⁺ T-cells co-cultured with BMDCs pre-incubated with 100 µg EVs isolated from the supernatants of ID8-ARG1 cells (EVs-ARG1) or ID8-pLVX (EVs-pLVX) cells for 3 days. EVs-ARG1 or EVs-pLVX with no BMDCs and/or ARG inhibitor OAT-1746 (200 nM) were added to some groups as indicated in the figure. d Relative CD3ε expression evaluated with flow cytometry in αCD3/αCD28-stimulated CD4⁺ (upper graph) and CD8⁺ (lower graph) T-cells after pre-incubation with EVs-ARG1 or EVs-pLVX (90 µg) and/or ARG inhibitor OAT-1746 (400 nM). Data show MFI of two technical repeats from n = 3 mice ± SD. e Representative proliferation histograms of SIINFEKL-specific CD8⁺ T-cells (OT-I T-cells) primed with SIINFEKL peptide pulsed BMDCs. Where indicated, DCs were pre-incubated with EVs-ARG1 or EVs-pLVX (100 µg) in the presence of 200 nM ARG inhibitor OAT-1746. Source data for panels b and d are provided as a Source Data file