Objective To evaluate T cell activation driven by exosomes from mouse lung epithelial MLE-12 cells (MLE-12 cells) irradiated with ⁶⁰Co γ ray.

Methods MLE-12 cells were divided into a control group and a ⁶⁰Co γ irradiation group (2, 4, 6, and 8 Gy), and exosomes were extracted from the supernatant of their culture medium by using ultracentrifugation. Nanoparticle tracking analysis and transmission electron microscope were used to determine the morphological structure and quantity of exosomes. The expression of lysosomal associated membrane protein (CD63), tetraspanin (CD81), tumor susceptibility gene (TSG101), and type Ⅰ endoplasmic reticulum protein (Calnexin) in exosomes were identified by Western blot (WB). Flow cytometry (FCM) was used to detect the expression of major histocompatibility complex class Ⅰ (MHC Ⅰ), major histocompatibility complex class Ⅱ (MHC Ⅱ), immune regulatory protein B7-1 (CD80), and immune regulatory protein B7-2 (CD86) on the surface of exosomes. Naive T cells isolated from mouse spleens were cocultured with exosomes (exo/NC MLE) secreted by MLE-12 cells in the control group (NC MLE-12) and exosomes (exo/IR MLE) secreted by MLE-12 cells in the 6 Gy ⁶⁰Co γ irradiation group (IR MLE-12), respectively. FCM was used to detect the changes of T cell subsets CD3⁺, CD4⁺, and CD8⁺ and their activated proliferation indicators T cell specific surface glycoprotein CD28 and early activation antigen 1 (CD69). Naive T cells were incubated with NC MLE-12, IR MLE-12, and MLE-12 cells from exosome inhibitor GW4869-treated groups, respectively. FCM was used to detect the changes of T cell subsets CD3⁺, CD4⁺, and CD8⁺ and their activation indicators CD28 and CD69. Independent samples t-test was used for comparison between two groups. Analysis of variance was used to compare multiple groups. Bonferroni adjustment was applied for pairwise comparison between two groups.

Results The exosomes produced from MLE-12 cells showed a typical saucer-like structure, with a particle size of 30–150 nm. WB results showed that the exosomes specific markers CD63, CD81, and TSG101 were highly expressed in exosomes, but the negative marker Calnexin was low in expression, compared with the MLE-12 cells. Compared with the control group, at different times after 6 Gy γ ray irradiation, the number of exosomes secreted by a single MLE-12 cell increased at 24 and 48 hours (t=5.36, 6.66, both P<0.05). The phenomenon of an increase in the number of exosomes secreted by a single MLE-12 cell 24 hours after irradiation with different doses of γ rays has a dose-effect relationship, and the difference is statistically significant at doses of 6 and 8 Gy (t=4.14, 5.67, both P<0.05) after the MLE-12 cells were irradiated with γ ray. The expression levels of MHC Ⅰ, MHC Ⅱ, CD81, and TSG101 increased in exo/IR-MLE compared with exo/NC-MLE. FCM results showed that the expression levels of MHC Ⅰ, MHC Ⅱ, CD80, and CD86 increased in exo/IR-MLE compared with exo/NC-MLE (t=4.04–6.47, all P<0.05). Compared with the exo/NC-MLE, in the T cells cocultured with exo/IR-MLE, the CD3⁺, CD4⁺, and CD8⁺ T cells all proliferated (t=3.08–5.88, all P<0.05), and the expression levels of CD28 and CD69 increased (t=3.02–8.65, all P<0.05). The exosome inhibitor GW4869 can suppress T cell activation induced by IR MLE-12 (t=3.64–23.03, all P<0.05).

Conclusion Exosomes from MLE-12 cells irradiated with ⁶⁰Co γ ray could activate T cells through antigen presentation.