*Purpose: New therapeutic approaches to the management of transplant recipients are warranted in efforts to improve outcomes.

*Methods: We have engineered a modified human IL-2 protein for preferential binding to the trimeric human IL-2 receptor over the corresponding dimeric IL-2 receptor. This was done by significantly reducing the affinity of IL-2 for CD122 while maintaining native affinity for CD25. In addition, the IL-2 mutein was fused to human IgG1 Fc to promote its extended half-life in vivo. We evaluated the ability of human IL-2 mutein as a tool to enhance Foxp3+ T-regulatory (Treg) cell functions in vitro and in murine allograft recipients.

*Results: In the presence of TGF-β, IL-2 mutein induced Stat5b phosphorylation and promoted inducible murine Treg developmentin vitro by 5-fold or by 8-fold when used in conjunction with a small molecule Cdk8/19 inhibitor that inhibits STAT5b dephosphorylation. Similarly, upon injection in vivo (10 µg s.c. twice/week), IL-2 mutein expanded the murine Foxp3+ Treg population by 4-5-fold. We therefore tested the effects of IL-2 mutein therapy in a fully MHC-mismatched cardiac allograft model (BALB/c->C57BL/6) in which recipients also received low-dose rapamycin (RPM, 0.5 mg/kg/d via Alzet pump for 2 wk post-Tx). Two injections of IL-2 mutein 10 µg s.c. twice in the week pre-Tx, plus low dose RPM for 2 weeks beginning at the time of transplant led to 80% long-term survival (>100 d, p<0.01 vs. RPM alone) but acutely rejected (7-8 d) third-party C3H allografts when challenged at >100 d. In a more clinically relevant protocol, the same dose of IL-2 mutein for 3 weeks post-Tx, in conjunction with low-dose RPM for 2 weeks, resulted in 100% survival for >100 d (p<0.01 vs. RPM alone or IL-2 mutein alone) and again was associated with the rapid (7-8 d) rejection of third-party C3H challenge allografts (p<0.01). Histologic examination of BALB/c allografts harvested at >100 d in mice treated with the IL-2 mutein/low-dose RPM protocol post-Tx showed excellent myocardial preservation, normal blood vessels, and only minor and focal mononuclear cell infiltrates. This contrasted with histologic evidence of ongoing graft rejection and areas of frank necrosis in allografts that maintained ventricular contraction to 100 days post-Tx in mice given 2 weeks of low-dose RPM and twice/week injections of IL-2 mutein for up to 80 days post-Tx.

*Conclusions: We conclude that a brief course of therapy with a selected IL-2 mutein can expand recipient Treg cells, without promoting Teff cell responses, and induce donor-specific allograft tolerance.

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