*Purpose: Recent studies demonstrated natural killer (NK) cells can induce cytotoxicity both by direct cytotoxic mechanisms and antibody (Ab) dependent cytotoxicity; however its role in the pathogenesis of chronic lung allograft dysfunction (CLAD) remains largely unknown. Exosomes released from NK cells have been shown to contain cytotoxic proteins (ie, FASL and perforin) with antitumor activities. The aim of the study is to immunologically and molecularly characterize exosomes isolated from mouse lung transplant recipients (LTxRs) with chronic rejection and elucidates the role of NK cells in eliciting immune response leading to CLAD.

*Methods: In the murine LTx model (unilateral left LTx performed between lungs of B6D2F1/J – a cross between C57BL/6J and DBA/2J – (Haplotype H2b/d) into DBA/2J (H2d) recipients), CLAD develops in >80% of animals within 30 days post-transplant. Bronchoalveolar lavage and serum were collected for Ab analysis by ELISA and isolation, characterization of circulating exosomes by Western blot using specific Abs. The lung sections were stained with trichrome, immune-histochemical (IHC) and morphometric characterization of NK cell infiltration using NKG2D marker.

*Results: In the murine LTx model, Abs to self-antigens (SAgs), Collagen V (Col-V) (>2.1 fold) and Kα1 Tubulin (Kα1T) (>2.8 fold) were elevated on day 14 when no histological evidence for CLAD was present and at the time of CLAD on day 28. Western blot results demonstrated increased levels with lung SAgs, Col-V (3.2±0.65vs 0.9±0.28; p=0.004), Kα1T (2.95±0.85 vs 0.75±0.15; p=0.011), NKG2D (2.8±0.25 vs 0.72±0.5; p=0.002), perforin (3.4±0.5 vs 1.72±0.45; p=0.012), and Fas L (3.5±1.2 vs 0.72±0.5; p=0.001) were also noted in circulating exosomes isolated from LTxRs with CLAD. Transplanted lung, but not native lung, also demonstrated increased cellular infiltration (CD3 (25%), CD4 (18%)), increased C4D deposition and fibrosis score (3.1±0.38). IHC analysis revealed increased NKG2D+ graft infiltrating cells (14% – day 28) as compared to native lung demonstrated a role for NK cells in the pathogenesis of CLAD.

*Conclusions: Circulating exosomes containing lung SAgs and NK cell related proteins were detected during CLAD in a murine model of chronic lung allograft rejection. We conclude that NK cell activation resulted in circulating exosomes with lung SAgs and NK markers can lead to immune stimulation resulting in de novo development of Abs to donor MHC and/or non-MHC lung SAgs leading to CLAD.

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