Abstract: Objective To isolate small extracellular vesicles(sEVs) derived from the supernatant of CD8⁺ regulatory T cells induced from human peripheral blood mononuclear cells (TR-sEVs), and explore their intervention effect and preliminary mechanism against transfusion-related acute lung injury (TRALI) in mouse model. Methods TR-sEVs were extracted by differential centrifugation combined with ultracentrifugation, and identified via nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and Western blotting (WB). A mouse model was established and grouped according to the two-hit hypothesis of TRALI. Mice received an intraperitoneal injection of lipopolysaccharide (LPS) as the first insult at 0 h. After 2 h, gradient doses of TR-sEVs were administered via tail vein, and anti-mouse H-2K^d antibody was injected via the tail vein at 24 h to establish the second insult. Mouse survival time and lung wet-dry weight ratio were recorded. Hematoxylin-eosin (HE) staining and immunohistochemical staining were performed to evaluate pulmonary pathological damage and inflammatory infiltration. The level of interleukin-6 (IL-6) in bronchoalveolar lavage fluid was detected by enzyme-linked immunosorbent assay (ELISA). An LPS-induced inflammatory injury model of EA.hy926 endothelial cells was established in vitro. Quantitative real-time polymerase chain reaction (qRT-PCR) and flow cytometry were used to detect the expression of intercellular adhesion molecule-1 (ICAM-1) in endothelial cells, and the adhesion effect of Tohoku Hospital Pediatrics-1 (THP-1) monocytes to endothelial cells was further evaluated. Results TR-sEVs increased the survival rate of TRALI mice, decreased lung wet-dry weight ratio (6.21±0.15 in control group vs 4.45±0.12 in intervention group), alleviated pulmonary inflammation and reduced IL-6 level. In vitro, TR-sEVs inhibited endothelial ICAM-1 expression and weakened monocyte-endothelial adhesion. Conclusion Infusion of TR-sEVs alleviates pulmonary lesions, inflammatory cell infiltration and the release of inflammatory cytokines in mice with transfusion-related acute lung injury. Its mechanism may exert effects via downregulating ICAM-1 expression in endothelial cells and suppressing monocyte-endothelial cell adhesion.

Key words: CD8⁺ Regulatory T Cells, Extracellular vesicles, Transfusion related acute lung injury, TRALI mouse model, Intercellular adhesion molecule-1, Monocyte-endothelial cell adhesion