Figure 7.

Exosomal αGC has comparable effects on NK cell activation, proliferation and missing self-function. C57Bl/6 mice were injected i.v., with PBS, 40 µg Exo(αGC-OVA) or soluble αGC (equivalent to the amount on 40 µg Exo(αGC-OVA) [47]. (A) Activation by CD69 and (B) KLRG1 expression was measured by flow cytometry on d7. (C,D) Mice were treated with PBS, 0.4, 4 or 40 µg exo(αGC-OVA) or with 200 ng soluble αGC and fed with BrdU in drinking water for 7 days. (C) Proliferation was assessed by measuring BrdU incorporation at day 7 on total NK cells. (D) NK cells were stained for inhibitory receptors (Ly49C/Ly49I/NKG2A/Ly49A/Ly49G₂) and the proliferation index was assessed as described in Materials and Methods. The proliferation index of the Ly49C⁺Ly49I⁺NKG2A⁺Ly49A⁻Ly49G₂⁻ (educated on self-MHC-I), the Ly49C⁻Ly49I⁻NKG2A⁻Ly49A⁺Ly49G₂⁺ (not educated on self-MHC-I) and the Ly49C⁻Ly49I⁻NKG2A⁻Ly49A⁻Ly49G₂⁻ (not educated on self-MHC-I) NK cell subsets are shown. (E,F) In vivo missing self-responses were measured by target cell elimination assay. (E) B6, MHC-I^−/− and CD1d^−/− mice were treated with 200 ng soluble αGC, 40 µg Exo(OVA) or 40 µg Exo(αGC-OVA) and challenged at day 7 with a mix of CFSE-labeled B6 (control) and β₂m^−/− spleen cells (target). The ratio of surviving target cells was examined after two additional days. (f) B6 mice were treated twice with 40µg exo(αGC-OVA) at an interval of 2 weeks. 3 days after the second treatment (stimulation 2), the rejection capacity was assessed by target cell elimination of CFSE-labeled B6 (control) and β₂m^−/− spleen cells (target). Data are pooled from 3 independent experiments (A–D) or from 2 independent experiments (E,F) with 7–10 mice per group (A,B), 3–10 mice per group (C), 4–10 mice per group (D), 4–12 mice per group (E) or 4–8 mice per group (F). Error bars denote SD. Significant differences were calculated with one-way ANOVA with Tukey’s correction for multiple testing and are denoted * p < 0.05, ** p < 0.01, *** p < 0.001.