*Purpose: The aim of the study is to determine the kinetics of induction of circulating exosomes with cardiac self-antigens (SAgs), cardiac myosin (MYO) and Vimentin (VIM), and their role in de novo development of Abs to SAgs leading to chronic rejection following heterotopic cardiac transplantation (HTx). Allogenic (BALB/c to C57BL/6) HTx was performed in the abdominal cavity. To induce tolerance leading to chronic cardiac allograft rejection, mice were treated with costimulatory blockade consisting of MR1 (250μg intraperitoneally [i.p.]) and CTLA4-Ig (200μg i.p.), on days 0 and 2, respectively. As reported by Shirasugi etal, these animals developed chronic rejection and it occurred between 40-45 days as evidenced histopathologically by myocyte damage, marked cellular infiltration with mononuclear cells and fibrosis.

*Methods: Sera were collected on days 7, 15, 30, & 45 for isolation and characterization of exosomes. Cardiac SAgs (MYO and VIM), Transcription factors, class II MHC trans-activator (CIITA), nuclear factor κB (NF κB) and 20S proteasome (20S) in exosomes were determined by western blot using specific Abs.

*Results: Circulating exosomes isolated from tolerance induced mice during chronic rejection on day 45 showed increased levels of SAgs MYO (4.7 vs 1.09), VIM (9.5 vs 1.35 p<0.01). More importantly, sera collected on days 15 and 30 demonstrated exosomes containing elevated levels of SAgs MYO (3.3 vs 1.09; p<0.05) & Vim (4 vs 1.35 p<0.01) respectively compared to control. CIITA was also increased on day 7, 15, & 30 (19.6, 29.4, & 8.7 vs 1.55; p<0.01) & day 45 (18.4; p<0.01) respectively. 20S proteasome levels were also elevated on day 30 & 45 (5.4 & 9.0 vs 1.49 p<0.01). NFκB levels in exosomes were also increased on day 7 (6.2 vs 1.2; p<0.01) and 15 (2.3 vs 1.2; p<0.05). Development of Abs to MYO occurred on day 15 (772±557 vs 187±50; p<0.01) and persisted on day 45 (1289±409 vs 187±50; p<0.001). Abs to VIM was also demonstrable in sera collected on day 45 (226 ±123 vs 89 ± 37; p<0.01). Wild type animals (Balb/c to C57BL/6) without induction of tolerance rejected hearts on day 7 to 8 (n=3 rejected/3 transplant) and sera demonstrated exosomes with cardiac SAgs, CIITA, NF κB and 20S proteasome and Abs to SAgs.

*Conclusions: We conclude that circulating exosomes containing SAgs (MYO and VIM), transcription factors (CIITA, NF κB), and 20S proteasome develop prior to development of antibodies to cardiac SAgs which we propose leads to the development of chronic rejection following allogenic HTx.

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