OR47 A novel role for antibodies to HLA and self-antigens: Induction of immuno-stimulatory exosomes

Abstract

Aim Recent report from us demonstrated that lung transplant recipients who developed donor specific antibodies (DSA) against HLA and chronic rejection (Bronchiolitis Obliterans Syndrome (BOS)) had circulating exosomes expressing donor HLA and lung self-antigens (SAgs), Collagen-V and K α 1-Tubulin, (AJT,17:474,2017). The goals of this study are to determine the role of DSA and antibodies (Abs) to SAgs in the development of exosomes and to define their immunogenicity. Methods Towards determining exosome development following ligation of antigens by Abs, an in vitro assay using human airway epithelial cells (AEC) incubated with allo-Abs to HLA (high panel reactive antibody sera) or Abs to lung SAgs was performed. After incubating AEC for 24 h, supernatants were collected for exosome isolation. Exosomes were isolated by ultracentrifugation and lack of aggregates determined using sucrose density gradient. Presence of exosome marker CD-9 and Alix, SAgs, costimulatory molecules (CD-80 & 86), HLA-class II and 20 s proteasome subunit α 3 were determined by immunoblot. Exosomes isolated from BOS patients were used for immunization of mice with or without adjuvants and immune responses determined, Abs by ELISA and SAg specific cytokines by ELISpot. Results Culture of AEC with either anti-HLA or Abs to SAgs, but not control, resulted in exosome secretion to the supernatant. Cells remained viable (>90%) at 24hrs when supernatant was collected for exosome isolation. Exosomes demonstrated markers CD-9 and Alix, lung SAgs, costimulatory molecules (CD80 & 86), HLA-class II and 20S proteasome. Immunization of mice with exosomes from BOS patients with or without adjuvant resulted in Abs to K α 1-Tubulin and Collagen-V. Splenic T cells responded to both SAgs with increased frequencies of IFN-’Y and IL17 with reduction in IL10. Conclusions Ligation of AEC by anti-HLA or anti-lung SAgs results in exosome development without cell death. Exosomes contain lung SAgs along with HLA-class II, 20S proteasome and costimulatory molecules making it highly immunogenic. Therefore, exosomes released from AEC upon binding by either de novo developed DSA or Abs to lung SAgs will augment immune responses following lung transplantation leading to rejection.