Background: It is generally accepted that antibody (Ab) binding to its specific antigen, in addition to activating complement pathway, can induce intracellular signals leading to rejection of allograft. Recent studies show that human lung transplant (LTx) recipients who developed Abs to donor human leukocyte antigens (HLA) had circulating exosomes containing lung associated self-antigens (SAgs). However, the ability of Abs binding to airway epithelial cells (AEC) in inducing exosomes from transplanted organ remains unknown. Using human AEC we analyzed the ability of Abs to HLA class-I and lung associated SAgs, K-alpha1Tubulin (Kα1T) and Collagen V (Col-V) to induce exosomes and determine their expression of SAgs and signaling molecules.

Methods: AEC were incubated for 24 hrs with Abs to lung SAgs, Kα1T or Col-V, HLA class-1 framework (W6/32), isotype control (C1.18.4), high panel reactive human sera to HLA and normal human serum (exosome depleted). Exosomes were isolated by ultracentrifugation and validated for exosome marker CD-9. Presence of SAgs, HLA class-I, co-stimulatory molecules (CD-80, CD-86), and nuclear transcription factor (hypoxia-inducible factor-1alpha (HIF-1α), nuclear factor-κB (NF-[Kappa]b)) were detected by immunoblot. Blocking of Ab induced exosome development from the AEC was done using GW4869 (20[micro]M) at the time of Ab incubation.

Results: AEC incubated with Abs (20[micro]g/ml) to SAgs, and HLA class-I (W6/32), but not isotype control, demonstrated induction of exosomes containing SAgs, donor HLA class-I, co-stimulatory molecules and NF-[Kappa]b. AEC treated with Abs to Kα1T not only induced exosomes containing Kα1T, but also Col-V. Similarly, AEC incubated with anti-Col-V produced exosomes containing not only Col-V but also Kα1T. High panel reactive Abs sera with Abs to HLA, when incubated with AEC, demonstrated exosomes containing SAgs, Col-V and Kα1T. In all of the above mentioned experiments, exosome formation in AEC was significantly reduced at concentration 20[micro]M of GW4869.

Conclusion: Abs binding to HLA class-1, or lung SAgs, induced exosomes releases in vitro from AEC. The released exosomes contained lung SAgs, co-stimulatory molecules (CD-80, CD-86) as well as HIF-1α and NF-κB suggesting that it will not only induce immune responses, but also may upregulate fibrinogenic growth factors involved in chronic rejection following human LTx.

CITATION INFORMATION: Gunasekaran M, Sharma M, Bansal S, Biswas Roy S, Walia R, Smith M, Bremner R, Mohanakumar T. Antibodies Specific for HLA Class I and Lung Associated Self-Antigens, Kα1Tubulin and Collagen V, Induces Exosomes Following In Vitro Binding to Human Airway Epithelial Cells. Am J Transplant. 2017;17 (suppl 3).

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