*Purpose: Antibodies (Abs) to human leukocyte antigen (HLA) (anti-HLA) can injure allograft by complement-dependent/independent mechanisms. Crosslinking of HLA-class I with Abs associate with integrin β4 and Src/FAK signaling pathway in endothelial cells and induction of endoplasmic reticulum (ER) stress. We sought to determine whether anti-HLA both class I and II can induce circulating exosomes from human airway epithelial cells in vitro and mechanism involved in exosome release.

*Methods: Human airway epithelial cell line (KCC266) was incubated with anti-HLA-A2 or high panel reactive sera (ie, anti-HLA), W6/32 (anti-HLA class I), anti-HLA class II (DR and DQ) for 24 hours. Exosomes were isolated by ultracentrifugation; size was defined by Nanosight NS300. Western blot was performed to determine lung self-antigens (Kα1Tubulin, Collagen V). Ability of anti-HLA to induce stress kinases and influence of exosome inhibitor GW4869 (20µM) in exosome release were determined.

*Results: Airway epithelial cells incubated with anti-HLA demonstrated induction of exosomes (<200 nm) containing self-antigen (Collagen V) (fold change: anti-A2, 3±1; anti-HLA, 2±0.5). Anti-HLA class I and II also induced exosomes with self-antigens (Collagen V (W6/32, 3±0.4; Abs to HLA-DR, 3±0.5; Abs to HLA-DQ, 2±0.6, and Kα1Tubulin (W6/32, 2±0.6; Abs to HLA-DR, 2.±0.4 Abs to HLA-DQ, 1.9±0.3). Anti-HLA induced ER stress markers in airway epithelial cells; PKR-like endoplasmic reticulum kinase (1.52±0.1) and eukaryotic translation initiation factor α (1.8±0.3). Anti-HLA class I (W6/32) also induced ER stress markers in airway epithelial cells; PKR-like endoplasmic reticulum kinase (2±0.8) and eukaryotic translation initiation factor α (2±0.4). Exosome release from airway epithelial cells was inhibited by GW4869 (20µM; 82±8%) but did not abrogate ER stress. Anti-HLA class I upregulated integrin β4 /FAK/SRC pathway and ER stress in AEC and lead to exosome release.

*Conclusions: Ligation of HLA molecules (class I and II HLA-DR, DQ) on human airway epithelial cell line by Abs specific to HLA induced exosomes with lung self-antigens (Kα1Tubulin, Collagen V), and ER stress in vitro. Anti-HLA class I ligation induced integrin β4 signaling by FAK/SRC pathway and ER stress which lead to release of exosomes from airway epithelial cells. We, therefore, present a new mechanism by which anti-HLA-induced stress of the transplanted organ can induce exosome release with self-antigens, contributing to the pathogenesis of rejection of the allograft.

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