Low Dose IL-2 Therapy Induces T Regulatory Cell Derived Circulatory Exosomes Containing PDL1 and CD73 and Abrogates Development of Chronic Cardiac Allograft Rejection

Abstract

<h3>Purpose</h3> Aim of the study is to determine the effect of low dose interleukin-2 (IL-2) therapy on the kinetics of induction of circulating exosomes and their immunological properties resulting in prevention of chronic rejection following allogeneic murine cardiac transplantation (HTx). <h3>Methods</h3> BALB/c (H2^d) to C57BL/6 (H2^b) HTx was performed heterotopically in the abdominal cavity. Costimulatory blockade consisting of MR1 (250 μg/ip day 0) and CTLA4-Ig (200 μg/ip day 2) was administered to induce tolerance. IL-2 (2000IU/day) was administered from day 14 post-transplant for 3 weeks. Sera were collected on days 7-100 for analysis of exosome induction and characterization of cardiac self-antigens (SAgs) (Myosin, Vimentin) by western blot. Foxp3, PDL1, CD73 in exosomes were detected using aldehyde bead - based flow cytometry. Antibodies (Abs) to donor MHC and SAgs were detected using immunofluorescence and ELISA respectively. <h3>Results</h3> BALB/c to C57BL/6 HTx acutely rejected (AR) with median survival time of 8 days. Costimulatory blockade prevented AR but developed chronic rejection with mean of 42 days circulating exosomes contained significantly higher levels of Myosin and Vimentin in the exosomes day 15 (p<0.05) and day 30 (p<0.01). Abs to MHC (32±13.8% vs 2±1.1; p<0.001) was detected on day 30, whereas Abs to Myosin (1114.21±443.4 ng/ml; p<0.0001) and Vimentin (519±241 ng/ml; p<0.0001) were detected on day45 during chronic rejection with fibrosis (57.1±20.7 vs 2.3±1.7%). Low dose IL-2 significantly prolonged the survival, 115 vs 42 (p<0.001) and did not induce exosomes with cardiac SAgs or developed Abs to MHC or SAgs. Low dose IL-2 not only abrogated chronic rejection but also induced splenic CD4+CD25+ Foxp3 T regulatory (Treg) cells in the allograft by day 45. Flow cytometry of circulatory exosomes isolated in day 45 also demonstrated increased Foxp3+ PDL1+ (3.3 fold) and FoxP3+ CD73+ (3 fold) compared to exosomes isolated on day 45 from chronic rejecting animals. <h3>Conclusion</h3> Our results demonstrate that low dose IL-2 abrogated the development of chronic rejection following HTx and induced circulating Treg cells derived exosomes with PDL1 and CD73 along with splenic Tregs and graft infiltrating FoxP3 cells.