Pre-transplant levels of autoantibodies to perlecan/LG3 predict poor allograft outcomes in renal transplant patients. We showed previously thatapoptotic exosome-like vesicles (ApoExo) enhance anti-LG3 production and rejection. The activity of the proteasome present within these vesicles is central to their immunogenic function. Here, we test the hypothesis that tissue injury (TI) enhances the release of ApoExo leading to increased circulating levels of ApoExo and anti-LG3 antibody production.

We used two murine models of TI, (acute or sustained) to test this hypothesis. Acute kidney injury (AKI) was induced with renal artery clamping and contralateral nephrectomy. The sustained model TI was evoked by femoral arteriectomy leading to hind-limb ischemia with progressive reperfusion over 3 weeks. Serum ApoExo were isolated by sequential centrifugation. The presence and activity of the proteasome within circulating ApoExo were evaluated respectively by electron microscopy with immunogold labelling and the Proteasome-GloTM Assay. LG3 levels were evaluated by Western Blot. Serum anti-LG3 IgG titers were monitored by ELISA.

In the acute TI model (AKI), the 20S proteasome was identifiedby immunogold-labellingand its activity (p<0.05) and LG3 levels (p<0.01) were significantly increased in circulating ApoExo preparations 2 days post-injury. Anti-LG3 IgG titers increased at 7 (p<0.05) and 14 days (p<0.001) post-surgery but returned to normal at 21 days. In the sustained TI model, circulating proteasome activity (p=0.01) and LG3 levels (p<0.01) also increased significantly in circulating ApoExo preparations 2 days post-surgery. This was associated with rising anti-LG3 IgG titers at 7 days post-surgery (p<0.05) but these remained elevated up to 21 days (p<0.01). As a control, non-injured mice injected i.v. every other day for 3 weeks with purified ApoExo also showed significantly increased anti-LG3 levels (p<0.001).

Collectively, these results show that autologous TIfavors the release of ApoExo known to trigger the production of anti-LG3 antibodies. These results suggest that tissue injury prior to transplantation, especially if sustained, represents a mechanism of importance in triggering the formation of anti-LG3 antibodies.

CITATION INFORMATION: Dieudé M, Turgeon J, Yang B, Pomerleau L, Hamelin K, Lan S, Qi S, Gingras D, Dhari W, Rivard A, Hébert M.-J. Exosome-Like Vesicles Released in Association with Tissue Injury Trigger Anti-LG3 Production. Am J Transplant. 2016;16 (suppl 3).

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