Introduction: Chronic antibody mediated rejection is one of the leading causes for late graft failure and until now, there is no specific treatment. It was shown that T-regulatory cells (Tregs) are crucial for suppression of auto- and alloimmune responses and that expansion of Tregs can induce immunological tolerance. Selective in vivo expansion of Tregs can be achieved with a specialspecific interleukin-2 (IL-2) anti-IL-2 treatment (IL-2 complexed to a specific antibody against IL-2 (JES6-1) = IL-2 cplx). This study aimed to induce long term heart allograft tolerance in mice that were treated with IL-2 cplx in combination with short-term low dose immunosuppressive therapy.
Methods: C57BL/6 mice were transplanted with a fully mismatched BALB/c heart under IL-2cplx, rapamycin and anti-IL-6 treatment. Recipient mice received IL-2/ α-IL2 (1 µg/5 µg) and Rapamycin [R1] (1 µg/g) on day -3, -2, -1 and three times a week after heart transplantation (HTX) until day 30. Anti-IL-6 was injected on day -1 (600 µg), 4 (300 µg) and 6 (300 µg). Cardiac allograft survival monitoring was performed using the palpation score. Loss of palpable heart beat was noted as day of rejection. The analysis of the Treg frequency in the blood during the follow-up period was measured by flow cytometry. Flow cytometry crossmatch (FCXM) was performed to detect donor specific antibodies (DSA). 
Results: Notably, indefinite survival was observed in the IL-2cplx protocol treatment group (>150 days), whereas the mean survival time (MST) in fully mismatched untreated recipients was only 7.5 days. Recently, our group tested this treatment protocol in a skin transplant model, where MST was 76 days, significantly less than in the HTX model. [R2] Of interest, Treg frequencies within CD4+ cells populations in the blood were markedly increased if compared to untreated heart graft recipients on day 21 post HTX (p=0.01) as well as day 28 post HTX (p=0.008) respectively. More importantly, on day 150, treated allograft recipients had significantly less DSA IgG1 (p=0.0002) and almost no IgG2ab (p= <0.0001) compared to untreated graft recipients. 
Conclusion: We could show that selective in vivo expansion of Tregs by IL-2 cplx, combined with Rapamycin and a short term anti-IL6 mAb treatment leads to operational tolerance in a clinically relevant and stringent model of cardiac transplantation. Moreover, recipient mice treated with IL-2 cplx presented with significantly reduced the levels of DSA even >100 days, suggesting induction of humoral tolerance. We suggest that our IL-2cplx-based treatment promotes immune tolerance in heart transplant recipients, as shown by indefinite survival in the absence of immunosuppressive treatment and the absence of DSA..[R3].