Fig. 6

Subcutaneously administered EVs transfer ARG1 to local lymph nodes and block OVA-antigen-specific T-cell proliferation. a Right inguinal lymph nodes from C57BL/6 mice were collected 4 or 24 h post EVs (250 µg) or PBS (Neg-ctrl) inoculation, lysed, and analyzed with immunoblotting for V5 tag. Pos-ctrl1: lysate of control lymph node with ex vivo added EVs-ARG1. Pos-ctrl2: lysate from ID8-ARG1-V5 cells. β-actin served as equal protein loading control. b–d OVA-specific, CTV-stained OT-I T-cells were injected i.v. into C57BL/6 mice and 24 h post adoptive transfer 5 μg of OVA protein 100 μg of EVs were injected daily for the consecutive 3 days s.c. into the right thigh. Where indicated, mice received 20 mg/kg of ARG inhibitor (OAT-1746) i.p. twice a day for the 3 following days. Each experimental group consisted of n = 4–9 mice. After 72 h CD8⁺ T-cells were isolated from right and left (control) inguinal lymph nodes, stained with SIINFEKL-H-2Kb tetramers and analyzed for proliferation. b Percentages (upper graph) and exemplary histograms (lower panel) of proliferating OT-I T cells isolated from mice immunized with ovalbumin (OVA) and injected with control EVs-pLVX or EVs-ARG1 EVs. T-cells isolated from the contralateral lymph node served as negative control (Ctrl LN). Representative experiment out of n = 2 is shown, data show means ± SD, P value was calculated with Kruskal–Wallis with Dunn’s multiple comparison test. c Percentages (upper) and exemplary histograms (lower panel) of proliferating OT-I T cells isolated from mice immunized with OVA and injected with EVs-ARG1 and/or ARG inhibitor OAT-1746. Representative experiment out of n = 2 with similar results is shown, data show means ± SD, P values were calculated with one-way ANOVA with Bonferroni post-hoc test. d Percentages of CD69⁺ T-cells (left), mean fluorescence intensity (MFI) for CD69 staining (middle) and MFI for CD3ε staining in OT-I T cells isolated from mice immunized with OVA and injected with EVs-ARG1 and/or ARG inhibitor OAT-1746. Representative experiment out of n = 2 is shown. Data show means ± SD, P values were calculated with Kruskal–Wallis with Dunn’s multiple comparison test. Source data for panels b–d are provided as a Source Data file